Tannin-chitosan composites

ABSTRACT

The invention provides a composition comprising a matrix of chitosan and a tannin wherein the chitosan is electrostatically bonded to the tannin to form a chitosan-tannin composite material. The chitosan can be partially or fully deacetylated, and the tannin can be a monomeric or an oligomeric proanthocyanidin or a hydrolysable tannin. The chitosan-tannin composite material can be a nanoparticle, a hydrogel film, a bio-foam, or a biogel, or the chitosan-tannin composite material can coat a liposome. The composite materials can be used for drug delivery, for antibacterial and/or antifungal applications, for tissue engineering applications, for wound healing applications, or they can be used as adjuvants for vaccination, including oral vaccinations. The invention also provides methods of preparing the composite materials and their various forms.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 61/240,033, filed Sep. 4, 2009, whichis incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. AT003846awarded by the National Institutes of Health. The United StatesGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Natural polymers have been used in many pharmaceutical applications andmedical device technologies. One natural polymer, chitosan, has beenused for the preparation of nanoparticles, microspheres, hydrogels,films, fibers, and tablets. Chitosan has been used to prepare potentialdrug delivery systems such as oral, nasal, parenteral, transdermal andophthalmic formulations. Chitosan has also been used to prepare wounddressings and tissue scaffolds (Kumar et al., Chem. Rev. 2004, 104,6017-6084). However, chitosan formulations and materials suffer fromnumerous drawbacks including limited stability, biodegradability andtensile strength. Materials such as modified chitosan and syntheticcomposite materials have tested for many of the same uses for whichchitosan has been evaluated but many of these materials suffer fromsimilar drawbacks, including insufficient biocompatibility.

Accordingly, there is a need for new materials that are biocompatibleand biodegradable, and that have suitable stability and mechanicalproperties for use in human and other mammalian treatments andtherapies. These new materials and compositions would preferably haveadvantages over chitosan alone, such as additional and/or improvedantimicrobial and antifungal properties, and improved physicalproperties. The ability to use these materials as tissue scaffoldsand/or systems for the delivery of therapeutic agents would further aidresearchers in the areas of biomaterials and drug delivery.

SUMMARY

The invention provides biodegradable and biocompatible tannin-chitosancomposites. The new composites can be formed into a variety of materialssuch as hydrogel films, three-dimensional foams, nanoparticles, andliposome coatings. The tannin-chitosan composite materials are strongerand have better mechanical properties than known chitosan materials. Thetannin component of the composite adds antifungal, antibacterial, andantioxidant properties to the antimicrobial property of the chitosancomponent, thereby significantly increasing the effectiveness of thecomposite in therapeutic applications.

The invention therefore provides a composition comprising a matrix ofchitosan and one or more tannins wherein the chitosan iselectrostatically bonded to the tannins to form a chitosan-tannincomposite material. The tannins can be at least dimeric in composition.For example, the tannins can be oligomeric proanthocyanidins oroligomeric hydrolysable tannins The compositions, or the tannins used toform the composition, can be substantially free or completely free ofmonomeric tannin components. The chitosan can have a deacetylationdegree of about 80% to about 99%. The mean molecular weight of thechitosan can be about 170 kDa to about 400 kDa. In some embodiments, themass of the tannins in the composite material can be about 1% to about50% of the mass of the chitosan in the composite material. In someembodiments, the mass of the tannins is about 5% to about 30% of themass of the chitosan.

By the selection of appropriate starting material (e.g., fruits, juice,presscake) and isolation procedures, the amount and proportion oftannins obtained with higher degrees of polymerization (DP) can becontrolled. For example, tannins with DPs of greater than 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, or 13 can be isolated and used in the compositionsdescribed herein. Additionally, the tannins isolated can be biased orcontrolled in terms of the nature of interflavan linkages (A-type vs.B-type), as well as their structural heterogeneity.

In one embodiment, the tannin can be a proanthocyanidin (PA). Theproanthocyanidin can have a degree of polymerization of at least, forexample, 4, 5, 6, or 7. The proanthocyanidin can have at least oneA-type interflavan bond. By the isolation processes described herein,proanthocyanidins with DPs of 2 to about 13, about 14 to about 20, about2 to about 20, or about 8 to about 20, can be selectively isolated.

In another embodiment, the tannin can be a hydrolysable tannin (HT). Insome embodiments, the hydrolysable tannin can include 2-5, 2-8, 2-10, or2-12 glucose units in its core structure. The hydrolysable tannin caninclude punicalagin. In some embodiments, the hydrolysable tannin caninclude at least about 80%, at least about 85%, at least about 90%, orat least about 95% punicalagin or another specific tannin describedherein.

The chitosan-tannin composition can be a nanoparticle, a hydrogel film,a bio-foam, or a biogel. Nanoparticles can be formulated into varioustherapeutic agent delivery systems, such as oral solutions, IVsolutions, or aerosols. Films can be formed into wound healing orpackaging materials. Foams can be used as scaffolds, such as for tissueengineering. The chitosan-tannin composition can also be used as asurface coating, for example, on the surface of a liposome.

The diameter of a chitosan-tannin composite material-coated liposome canbe about 300 nm to about 800 nm. The chitosan-tannin composite materialcan form a nanoparticle that has a diameter of about 75 nm to about 450nm, or about 100 nm to about 350 nm. In such embodiments, thenanoparticle can protect an encapsulated material in variousenvironments, such as in the acidic conditions of the stomach. In someembodiments, the chitosan-tannin composite material can be crystalline.

The ratio of chitosan to tannins can be varied, in certain embodiments,from about 5:1 to about 1:5. The composite material can be optionallycrosslinked. The amount of crosslinking agent used in preparingnanoparticles, with respect to the mass of chitosan, can be about 1:4 toabout 1:25. Crosslinking agents that can be used includetripolyphosphate (TPP) anions, glutaraldehyde, glyoxal, sucrose, and thelike.

The invention also provides a method for delivering a bioactive agent toa mammal comprising administering to a mammal a chitosan-tannincomposite material described herein. The chitosan-tannin compositematerial can form a nanoparticle that encapsulates the bioactive agent,for example, a drug or nutrient. Examples of drugs and nutrients includelipids such as fatty acids, including omega-3 and omega-6 fatty acids,fat soluble vitamins (e.g., vitamin A, D, E, and/or K), antibiotics,probiotics, micronutrients such as β-carotene and/or ascorbic acid,proteins, and peptides. In some embodiments, monomeric tannins and othernutritional supplements can also be included in a chitosan-tannincomposite matrix, or in a composition that includes a chitosan-tannincomposite matrix.

The invention further provides methods to inhibit bacterial growth orfungi growth in an animal, a plant, food, or in vitro. The methods caninclude treatment of the animal, plant, or food, prophylactically orafter colonization by bacteria or fungi, by administering an effectiveamount of a chitosan-tannin composite material described herein, whereinthe composite material inhibits the bacterial growth or fungi growth.The bacteria or fungus can be any genera or species known to infectanimals, plants, or food. The bacteria can be gram positive or gramnegative bacteria. Examples of bacteria that can be killed or whosegrowth can be inhibited include bacteria of the genera Escherichia,Erwinia, or Xanthomonas, for example, Escherichia coli, Erwiniacarotovora or Xanthomonas spp. Examples of fungi that can be killed orwhose growth can be inhibited include fungus of the genera Bothytis,Fusarium, or Colletotrichum, for example, Bothytis cines or Fusariumoxysporum, or Colletotrichum acutatum.

The invention additionally provides an adjuvant for oral vaccinationthat includes a chitosan-tannin composite material as described herein,such as a nanoparticle, and an antigen. The adjuvant can be a protein, apeptide, a nucleic acid, or DNA. The antigen can be encapsulated in thenanoparticle or adsorbed to the surface of the nanoparticle. Forexample, the composite material can encapsulate the antigen andgradually release it under physiological conditions. The compositematerial can therefore protect an encapsulated antigen to allow for oraldelivery. The composite material can also facilitate uptake by M cellsin the nasal-associated lymphoid tissue (NALT) when administered nasallyor by the M cells of the gut-associated lymphoid tissue (GALT) whenadministered orally, thus providing a vehicle for mucosal immunization.

The chitosan-tannin composite materials can be tailored to degrade overa range of rates under various conditions by varying the amounts of thecomponents and methods for preparing the composite materials. Thus, theinvention also provides a method of preparing a chitosan-tannincomposite material. The invention further provides for the use of acomposition described herein for the manufacture of medicaments usefulfor the treatment of conditions such as bacterial infection and/orfungal infection in a mammal, such as a human.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are includedto further demonstrate certain embodiments or various aspects of theinvention. In some instances, embodiments of the invention can be bestunderstood by referring to the accompanying drawings in combination withthe detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention, however, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIG. 1 illustrates a Matrix Assisted Laser Desorption/IonizationTime-of-Flight Mass Spectrometry (MALDI-TOF MS) spectrum in positivereflectron mode for the cranberry PA fraction. The section between m/z1000 and 2500 has been amplified to identify the degree ofpolymerization, the nature of interflavan linkage and structuralcomposition of subunits of oligomeric PA.

FIG. 2 illustrates a MALDI-TOF MS spectrum, in negative reflectron mode,of a pomegranate hydrolysable tannin.

FIG. 3 illustrates dissolving and degassing a chitosan solution,according to an embodiment.

FIG. 4 illustrates the preparation of the chitosan-tannin bioconjugates,according to an embodiment.

FIG. 5 illustrates the casting of the chitosan-tannin hydrogels,according to an embodiment.

FIG. 6 illustrates the preparation of chitosan-tannin compositenanoparticles, according to an embodiment.

FIG. 7 illustrates the preparation of chitosan-tannin bio-foams,according to an embodiment.

FIG. 8 illustrates the preparation of chitosan-tannin bio-gels,according to an embodiment.

FIG. 9 illustrates X-ray diffraction patterns of chitosan andtannin-chitosan composites. Arrows indicate the most appreciable changesgenerated by the tannin component on the crystal arrangement of thebiopolymer.

FIG. 10 illustrates DSC thermograms of chitosan (dashed line) andtannin-chitosan composites (solid line). FIG. 10A illustrates HT-derivedcomposites and FIG. 10B illustrates PA-derived composites. Arrowsindicate significant changes on the thermal behavior of the compositematerial.

FIG. 11 illustrates the results of mechanical behavior tests ofchitosan-tannin hydrogels compared to a chitosan hydrogel. Chito-PAC isa chitosan-proanthocyanidin composite; Chito-Pun is achitosan-punicalagin composite. Data is shown as [mean±SD; n=5].

FIG. 12 illustrates the degree of swelling in water for a chitosanhydrogen (Chto) and chitosan-tannin hydrogels (Ch-PAC and Ch-Pun) as afunction of time.

FIG. 13 illustrates scanning electron microscopy (SEM) micrographs forchitosan and chitosan-tannin nanoparticles: 13A: Chitosan Nanoparticles(ChNp); 13B: Chitosan Proanthocyanidin Nanoparticles (ChPANp); and 13C:Chitosan Hydrolysable Tannin Nanoparticles (ChHTNp). Scale bar indicates200 nm.

FIG. 14 illustrates the antimicrobial susceptibility test oftannin-chitosan composites against common Costa Rican agriculturalpathogens, according to certain embodiments. Data is shown as mean±SD;n=3. CNp (left bar for each bar group) refers to chitosan nanoparticles;CPACNp 10% (middle bar) refers to chitosan-proanthocyanidinnanoparticles (90 wt. % chitosan, 10 wt. % cranberry tannin; and CHTNp10% (right bar) refers to chitosan-hydrolysable tannin nanoparticles (90wt. % chitosan, 10 wt. % hydrolysable tannin.

FIG. 15 illustrates the influence of addition of chitosan-tannin (0 to0.5, w/v %) to liposomes (lecithin 0.4% w/v, 200 nm) on the ζ-Potentialvalues of the vesicles.

FIG. 16 illustrates the influence of the addition of chitosan-tannin (0to 0.5, w/v %) to liposomes (lecithin 0.4% w/v, 200 nm) on the meanparticle diameter of the vesicles.

FIG. 17 illustrates the proposed mechanism of action for the addition ofchitosan-tannin complexes to liposomes leading to coacervation, stablesecondary liposomal dispersions, and bridging flocculation.

FIG. 18 illustrates differential scanning calorimetry thermograms forCNp and CTNp.

FIG. 19 illustrates bovine serum albumin release profiles from CNp andCTNp. Results are shown as mean±standard error, n=3.

FIG. 20 illustrates positive mode MALDI-TOF MS [M+Na]⁺ of cranberryproanthocyanidins (PAC) from juice (top spectrum) and presscake (bottomspectrum). The juice PAC are oligomers from 2 to 7 degrees ofpolymerization (DP), whereas the presscake PAC are oligomers from 2 to13 DP in the main spectra and from 14 to 20 DP (m/z=5788) in the insert.

FIG. 21 illustrates the reduction in Colony Forming Units (CFU) ofCaco-2 E. coli after treatments with chitosan, cranberry presscake (CPC)tannins, and a chitosan-CPC composite, compared to a control experiment.

FIG. 22 illustrates the change in nanoparticle particle size as afunction of tripolyphosphate (TPP) concentration, for certain ratios ofspecific tannin-chitosan composites (see Table 7-2).

FIG. 23 illustrates the change in nanoparticle particle size as afunction of TPP concentration, for certain ratios of specifictannin-chitosan composites (see Table 7-3).

FIG. 24 illustrates the change in nanoparticle particle size as afunction of TPP concentration, for certain ratios of specifictannin-chitosan composites (see Table 7-4).

FIG. 25 illustrates the grape seed extract (GSE)/chitosan nanoparticlesize distribution, as determined by the procedures of Example 8.

FIG. 26 illustrates a dose-response curve, as determined in Example 8,for the effects of grape seed extract (GSE) and chitosan-GSE compositeson bacterial invasion of Caco-2 cells.

FIG. 27 illustrates relative invasion of gastrointestinal (GI)epithelial cells (Caco-2) by uropathogenic Escherichia coli (UPEC),according to the methods of Example 8. Thirty strains of UPEC isolatedfrom women with chronic UTI. The most invasive strain was UPEC 5011(black bar), which was selected for evaluation in invasion experiments.

FIG. 28 illustrates the effects (mean±SD) of cranberry tannins, andcranberry tannin-chitosan composite nanoparticles on the invasion ofCaco-2 cells by E. coli strain 5011. An asterisk indicates an effectthat is statistically significant compared to the corresponding control.

FIG. 29 illustrates the effects (mean±SD) of chitosan nanoparticles,cranberry tannins, and cranberry tannin-chitosan composite nanoparticleson the invasion of Caco-2 cells by E. coli strain 5011. An asteriskindicates an effect that is statistically significant compared to thecorresponding control. Dose was 0.75 μg gallic acid equivalent (GAE).

FIG. 30 illustrates Scanning Electron Microscopy (SEM) exploring theinteractions of cranberry proanthocyanidin-chitosan compositenanoparticles with UPEC 5011 and its effect on cell invasion. A) UPEC5011 alone (arrow indicates flagella); B) UPEC 5011+chitosannanoparticle (arrow indicates no change to flagella); C) UPEC5011+tannin-chitosan composite nanoparticle (arrow indicates coating andcrosslinking of UPEC flagella); and D) tannin-chitosan compositenanoparticles alone (for comparison to material coating flagella).

DETAILED DESCRIPTION

The invention provides new biodegradable, biocompatible compositematerials comprising a combination of chitosan and tannins Thechitosan-tannin composites are extremely versatile and can be formulatedinto various kinds of biomaterials, such as dermal patches,three-dimensional sponges for drug delivery and wound healing,biodegradable sutures, and scaffolds for cell proliferation in tissueengineering, as well as nanoparticles and liposomes for sustained drugdelivery.

The chitosan-tannin composites can also be used in the formulation ofvaccine antigens, including proteins, peptides, DNA, and the like. Inthese formulations, the antigen can be either entrapped or adsorbed ontothe surface of the particles. The particles can also be tailored todegrade over a range of times, at various rates. They can therefore actas a depot from which the encapsulated antigen is gradually released.Additionally, chitosan-tannin nanoparticles can offer protection toencapsulated antigens delivered orally, as well as facilitate uptake byM cells in the nasal-associated lymphoid tissue (NALT) when administerednasally and M cells in the gut-associated lymphoid tissue (GALT) whenadministered orally, thus serving as a vehicle for mucosal immunization.Accordingly, chitosan-tannin complexes can be used in various importantfields, such as environmental, drug delivery, tissue engineering, andother biomedical application.

Definitions

As used herein, certain terms have the following meanings. All otherterms and phrases used in this specification have their ordinarymeanings as one of skill in the art would understand. Such ordinarymeanings may be obtained by reference to technical dictionaries, such asHawley's Condensed Chemical Dictionary 14^(th) Edition, by R. J. Lewis,John Wiley & Sons, New York, N.Y., 2001.

References in the specification to “one embodiment”, “an embodiment”,“an example embodiment”, etc., indicate that the embodiment describedmay include a particular aspect, feature, structure, moiety, orcharacteristic, but not every embodiment necessarily includes thataspect, feature, structure, moiety, or characteristic. Moreover, suchphrases may, but do not necessarily, refer to the same embodimentreferred to in other portions of the specification. Further, when aparticular aspect, feature, structure, moiety, or characteristic isdescribed in connection with an embodiment, it is within the knowledgeof one skilled in the art to affect such aspect, feature, structure,moiety, or characteristic in connection with other embodiments, whetheror not explicitly described.

The term “and/or” means any one of the items, any combination of theitems, or all of the items with which this term is associated.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a compound” includes a plurality of such compounds, so that acompound X includes a plurality of compounds X.

The term “about” can refer to a variation of ±5%, ±10%, ±20%, or ±25% ofthe value specified. For example, “about 50” percent can in someembodiments carry a variation from 45 to 55 percent. For integer ranges,the term “about” can include one or two integers greater than and/orless than a recited integer. Unless indicated otherwise herein, the term“about” is intended to include values, e.g., weight percents, proximateto the recited range that are equivalent in terms of the functionalityof the individual ingredient, the composition, or the embodiment. Inaddition, a recited range (e.g., weight percents, carbon groups, and thelike) includes each specific value, integer, decimal, or identity withinthe range. Specific values listed herein for ranges and the like are forillustration only; they do not exclude other defined values or othervalues within defined ranges.

The phrase “one or more” is readily understood by one of skill in theart, particularly when read in context of its usage. For example, one ormore substituents on a phenyl ring refers to one to five, or one to upto four, for example if the phenyl ring is disubstituted.

The term “contacting” refers to the act of touching, making contact, orof bringing to immediate or close proximity, including at the molecularlevel, for example, to bring about a chemical reaction or physicalchange, e.g., in a solution or other reaction mixture.

An “effective amount” generally means an amount which provides thedesired effect. An effective amount therefore means a dosage sufficientto enhance the efficacy of treatment for a disease state or conditionbeing treated. Thus the effective amount can vary depending on thepatient, the disease, and the treatment being effected.

The term “patient” or “subject” refers to any animal, such as a mammal,including mice, rats, other rodents, rabbits, dogs, cats, swine, cattle,sheep, horses, or primates, and humans.

In reference to tannins, the phrase “substantially free of monomericcomponents” means that the tannins are at least dimeric in compositionand few or no monomeric tannin species are present in the sample. Forexample, a tannin that is substantially free of monomeric components caninclude less than about 5 wt. % monomeric tannins, less than about 3 wt.% monomeric tannins, less than about 1 wt. % monomeric tannins, lessthan about 0.5 wt. % monomeric tannins, less than about 0.1 wt. %monomeric tannins, or no monomeric tannins. An example of a monomerictannin is the compound tannic acid (pentagalloyl-D-glucose (C₇₆H₅₂O₄₆,mw=1701.18)). Other compounds that can be specifically included orexcluded from the composites described herein include catechin(mw=290.26), quercetin (mw=302.24), cyanidin (mw=287.24), gelatin,epchlorohydrin moieties, or combinations thereof.

Tannins.

Tannins include oligomeric polyphenols that occur naturally in a varietyof plants. Isolated tannins typically form a heterogeneous mixture oftannin compounds. Tannin compounds can be subdivided into two groups:condensed tannins, also known as proanthocyanidins (“PA” or “PAC”), andhydrolysable tannins (HT). Tannin oligomers typically occur as dimers,trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers,or decamers. Oligomers with greater than ten monomeric segments can alsobe isolated, such as oligomers that include up to 50 units, as describedherein. For a review of tannin nomenclature, see Beecher (J. Nutrition2003, 3248S-3254S), which is incorporated herein by reference. In someembodiments, certain monomeric tannins or low DP tannins can be excludedfrom a particular composition. For example, a composition may excludecatechin, tannic acid, or other monomeric tannins, dimeric tannins,trimers, or tetramers, PA tannins, or alternatively, HT tannins, acertain molecular weight range of tannins, or a type, class, or specifictannin cited in Beecher.

Proanthocyanidins are polymers of flavan-3-ols and flavans linkedthrough an interflavan bond between carbon 4 of the C ring and carbon 8of the A ring, as shown in Scheme 1. Scheme 1 illustrates a cranberrypolyflavan-3-ol showing structural variation in the nature ofinterflavan linkage and substitution to an anthocyanin terminal unitthrough a CH₃—CH bridge.

Scheme 2 illustrates two other types of condensed tannins (PAs):procyanidins and prodelphinidins (for the trimer x=1; for the tetramer,x=2; for the pentamer, x=3; for the hexamer, x=4; for the heptamer, x=5;for the octamer, x=6; for the nonamer, x=7; and for the decamer, x=8).Procyanidins (R═H) contain catechin and/or epicatechin (CE) subunits;prodelphinidins (R═OH) contain gallocatechin and/or epigallocatchin (GE)subunits.

In various proanthocyanidins, the R groups of Scheme 2 can eachindependently be H or OH. In some embodiments, one or more hydroxylgroups may be glycosylated. In some embodiments, x is 1 to about 50, 1to about 25, 1 to about 20, 1 to about 12, 1 to about 10, or a range ofbetween any to integers from 1 to 50. The condensed tannins (PAs) canhave various interflavanoid linkages (such as A-type 4→8 or 4→6interflavan bonds, or B-type 4→8, 2→O-7 interflavan bonds, each α or β),cis- or trans-stereochemistry, and one or more hydroxyl groups canoptionally be absent on the A-ring, B-ring, C-ring, or a combinationthereof.

Other PA tannins include glycosylated heteropolyflavans, such as thoseillustrated in Scheme 3. Representative compounds shown in Scheme 3include proluteolinidin (R′═OH); proapigininidin (R¹═H); eriodictyol(R²═H); and eriodictyol 5-O-β glucoside (R²=glucose). Krueger et al.have described a variety of known heteropolyflavans-3-ols andglycosylated heteropolyflavans (see J. Agric. Food Chem. 2003, 51,538-543, which is incorporated herein by reference).

where R¹ is H or OH; R² is H or glucose; and glu is glucose (e.g., aβ-glucoside).

In some embodiments, x of Scheme 3 is 1 to about 50, 1 to about 25, 1 toabout 20, 1 to about 12, 1 to about 10, or a range of between any tointegers from 1 to 50. Several examples of condensed tannins aredescribed in U.S. Pat. No. 7,122,574 (Romanczyk et al.), which isincorporated herein by reference.

A review by Reed et al. (Phytochem. 66(18): 2248-2263 (2005)) describesthe structural heterogeneity of tannin polyphenols from cranberries,grape seed extracts, sorghum, and pomegranates as characterized byMALDI-TOF MS. Examples of plants that produce proanthocyanidins includecranberries, blueberries, grapes, sorghum, and pine.

Hydrolysable tannins include gallic acid and ellagic acid esters ofpolyol core moieties, such as sugars. Scheme 4 illustrates a pomegranateellagitannin showing structural variation in nature of esterification ofthe glucose core molecule.

Hydrolysable tannins, such as the compound shown in Scheme 4, can beisolated in oligomeric forms that include 2 to about 12 hydrolysabletannin moieties, for example, linked by oxidative C—O coupling betweengalloyl and hexahydroxydiphenoyl moieties of the monomeric precursors.Common coupling also occurs between two ellagic acid moieties, or byaddition of gallic acid moieties to the saccharide core of an oligomer.See Quideau and Feldman, Chem. Rev. 1996, 96, 475-503, which isincorporated herein in its entirety.

Accordingly, in some embodiments of compositions described herein, thehydrolysable tannins employed will be oligomeric hydrolysable tanninsThus in some embodiments, oligomeric hydrolysable tannins include atleast two saccharide core moieties. Other embodiments can includemonomeric proanthocyanidins or hydrolysable tannins, and yet otherembodiments can exclude monomeric tannins. In some embodiments, ahydrolysable tannin will include one or more (e.g., 1, 2, 3, 4, 5, ormore) ellagic acid moieties, and in some embodiments, a hydrolysabletannin will include one or more (e.g., 1, 2, 3, 4, 5, or more) gallagicacid moieties.

Examples of plants that produce hydrolysable tannins includepomegranates, strawberries, raspberries, blackberries, and sumac.Significant quantities of hydrolysable tannins can be isolated from, forexample, pomegranate husks. Specific hydrolysable tannins includepunicalin and punicalagin (the alpha or beta isomer of2,3-(S)-hexahydroxydiphenoyl-4,6-(S,S)-gallagyl-D-glucose, with amolecular weight of 1084) and stereochemical isomers thereof, as well asthe hydrolysable tannins described by Quideau and Feldman (Chem. Rev.1996, 96, 475-503).

Consumption of foods, beverages and nutritional supplements that containtannins is associated with decreased risk of diseases that have anoxidative and microbial adherence etiology. Numerous studies show thattannins have various types of pharmacological properties includinganti-oxidative, anti-mutagenic, anti-carcinogenic, anti-angiogenic,apoptotic, anti-obesity, hypocholesterolemic, anti-arteriosclerotic,anti-diabetic, anti-bacterial, anti-viral, and anti-aging effects, aswell as wound healing properties.

Chitosan.

Chitin is a biopolymer composed of poly N-acetyl glucosamine. Chitin isthe second most abundant biopolymer on earth, after only cellulose. Itis commonly found in the exoskeleton or cuticles of many invertebrates,such as the shells of marine arthropods, and in the cell wall of mostfungi and some algae. Chitin is generally insoluble in water but can bedeacetylated by treatment with a caustic, such as sodium hydroxide, toform the soluble cationic polysaccharide, chitosan. The chemical name ofchitosan is poly(β-(1→4)-2-amino-2-deoxy-D-glucopyranose). Chitosan hastwo types of reactive groups that can be grafted: the free amine groupson deacetylated units, and the hydroxyl groups on the C3 and C6 carbonson either acetylated or deacetylated units (Scheme 5).

Chitosan is commonly used in water processing and in agriculture.Chitosan can also form a polycationic, biodegradable, and biocompatiblematrix with blood clotting and antimicrobial properties. Kumar et al.(Chemical Reviews (2004) 104:6017-6084) and Rinaudo (Progress in PolymerScience (2006) 31:603-632) have reviewed the properties and applicationsof chitosan. Due to its unique polycationic nature, chitosan and itsderivatives have been used for various applications in many differentfields, including biomedicine, food, agriculture, biotechnology andpharmaceutics. Chitosan has been developed for a variety of biomedicalapplications including wound dressings and drug delivery systems. In thecontext of drug delivery, chitosan has been used as a stabilizingconstituent of liposomes; as an excipient controlling drug release inoral formulations; as a nasal delivery system; to prepare microspheresfor encapsulation of enzymes, proteins and cells, and also to deliverDNA.

Research has shown that the water solubility, antibacterial, andantioxidant properties of chitosan can be improved by primary derivationfollowed by graft modification. Grafting chitosan is also a common wayto improve other properties such as increased chelating or complexationproperties, bacteriostatic effects, or enhanced adsorption properties.Although the grafting of chitosan modifies its properties, usefulcharacteristics such as mucoadhesivity, biocompatibility, andbiodegradability can still be maintained.

Chitosan-based bandages and surgical dressings produced by HemConMedical Technologies were recently approved by the U.S. FDA for use ashemostatic bandages with proven antibacterial properties against a widerange of harmful organism, including MRSA and acinetobacter baumannii.The bandages and dressings can be used to rapidly stop bleeding,including extensive arterial bleeding. Both the blood clotting and theantibacterial properties of the materials can be attributed to chitosan(see U.S. Pat. No. 7,482,503 (Gregory, et al.), which is incorporatedherein by reference). The tannin-chitosan composite material can be usedin place of the chitosan in the compositions described therein.

Chitosan is commercially available from many chemical suppliers, such asSigma Aldrich Co., St. Louis, Mo. Chitosan is offered in various grades,average molecular weights, and degrees of deacetylation.

In some embodiments, the chitosan can be a “high molecular weight”chitosan. High molecular weight chitosan refers to chitosan that has anumber average molecular weight of at least about 100 kDa, and typicallyabout 170 kDa to about 400 kDa. In some embodiments, high molecularweight chitosan can have a molecular weight of at least about 100 kDa,at least about 110 kDa, at least about 150 kDa, or at least about 200kDa. In other embodiments, high molecular weight chitosan can have amolecular weight of about 100 kDa to about 400 kDa, about 120 kDa toabout 400 kDa, about 150 kDa to about 400 kDa, about 170 kDa to about400 kDa, 100 kDa to about 300 kDa, about 120 kDa to about 300 kDa, about150 kDa to about 300 kDa, about 170 kDa to about 300 kDa. The value of nin Scheme 5 can be any number or range that results in approximately thevalues for the molecular weights of chitosan described herein. As wouldbe readily recognized by one of skill in the art, chitosan asillustrated in Scheme 5 may also be partially acetylated.

Other embodiments may include low molecular weight chitosan. Lowmolecular weight chitosan refers to chitosan molecules with less than100 monomeric units (less than about 18 kDa or less than about 20 kDa).Molecular weights of chitosan can be determined, for example, by gelpermeation chromatography.

The chitosan can have a degree of deacetylation that is typically atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99%, or thechitosan can be substantially fully deacetylated.

Tannin-Chitosan Composite Materials.

Chitosan binds to negatively charged tannins by electrostaticinteractions, driven by its positively charged amino groups. Thisinteraction allows for developing stable biomaterials, such asnanoparticles that can serve as therapeutic agents, as a targetedcarrier compositions, and controlled release system. The tannin-chitosancomposite biomaterial can also form biofilms, biofoams, and tissuescaffolds. Tannin-chitosan composite materials can be used to preparedermal patches, for example, for wound healing applications. Thetannin-chitosan composite materials can also be used as scaffolding forskin and cartilage tissue cultures, for matrices for tissue engineering,for coatings for biomedical devices, or as packaging materials, forexample, to prevent oxidation of organic materials such as meats and/orlipids, and/or for drug delivery.

The tannin-chitosan composite materials provide higher biocompatibilityand superior biodegradability relative to known synthetic composites. Inaddition to direct biological effects from the tannin-chitosancomposites (antimicrobial and antioxidant properties), the biomaterialscan be used for targeting or controlled release systems to encapsulatetherapeutic agents for oral, dermal, or respiratory delivery.

With respect to oral delivery, some studies have shown that theantioxidant activity of catechin, a component of tannins, decreasesdramatically when it is exposed to an alkaline pH, such as in the humanintestine. Considering the function of tannins in metabolic processes,as well as the properties of tannins manifested in vitro, complexationto chitosan can protect tannins from interactions with the food matrix,and can also provide for delayed or controlled release in thegastrointestinal tract.

Nanoparticles and biofilms can be prepared from chitosan and tannins, asdescribed herein. High molecular weight tannins are particularly usefulfor preparing nanoparticles. Low molecular weight tannins are wellsuited for preparing chitosan-tannin composite foams, whereas tannins ofintermediate molecular weight provide superior biofilms compared toknown chitosan biofilms. According to the specifications provided bySigma-Aldrich, a chitosan with low molecular weight refers to chitosanpolymer that has about 50 to about 250 monomeric units (5,000-10,000Da), a medium molecular weight chitosan sample has a molecular weight inthe approximate range of about 10,000 to about 100,000 Da. A highmolecular weight chitosan typically refers to a chitosan with amolecular weight of about 100,000 to about 500,000 Da.

Two classes of tannin-chitosan composite nanoparticles are theproanthocyanidin-chitosan composites and the hydrolysabletannin-chitosan composites. The proanthocyanidin-chitosan composites canbe prepared from, for example, cranberry tannins and chitosan. Theproanthocyanidins can also be obtained from, for example, grapes (e.g.,from fruit, wine, juice, or presscake), cinnamon, apples, or grainsorghum. The hydrolysable tannin-chitosan composites can be preparedfrom, for example, pomegranate tannins and chitosan. Other hydrolysabletannins can be obtained from, for example, strawberries, raspberries, orsumac.

The chitosan-tannin composite can be crosslinked during its preparation.Nanoparticles can be crosslinked, for example, with tripolyphosphate(TPP) anions. Biofilms can be crosslinked, for example, withglutaraldehyde. Swelling of a biopolymeric matrix can produce fasterdegradation/active compound release from the matrix. In both thenanoparticles and the biofilms, crosslinking reduces or preventsspontaneous swelling of the biopolymeric matrix. In nanoparticles, TPPstrengthens the polymeric networks to maintain a spherical nanoparticleshape. In biofilms, crosslinking can reduce or prevent fastrelease/degradation of the biofilm. By controlling the concentration ofthe crosslinker added during preparation, it is possible to control therelease rate of active compounds from the matrix. Additionally,increasing degree of polymerization of tannin component increasescrosslinking efficiency, thereby reducing amount of TPP needed for asimilarly stable particle, as well as influencing particle size. The DPcan be controlled by choice of starting material and chromatographicseparation, all of which can be monitored by MALDI MS.

In some embodiments, the chitosan-tannin composite material can includeor exclude polymers other than chitosan or tannins For example, someembodiments include cellulose or collagen-derived materials such asgelatin; other embodiments exclude them. Some embodiments includesynthetic polymers such as polyethylene or polyethylene oxide, whileother embodiments exclude them.

Antimicrobial Applications and Drug Delivery.

Chitosan itself has valuable biopharmaceutical characteristics such aspH sensitivity, biocompatibility and low toxicity. Chitosan is alsometabolized by certain human enzymes, especially lysozyme, and istherefore biodegradable. For drug delivery applications, it is importantfor the chitosan formulation to be hydro-soluble and positively charged.These properties enable it to interact with negatively charged polymers,macromolecules and polyanions in an aqueous environment.

The chitosan-tannin complexes described herein have higher protein anddrug loading efficacy and capacity, as well as better releaseproperties, than chitosan itself. The complexation of chitosan totannins enhanced its stability at different pH environments compared tochitosan alone. The chitosan-tannin complexes also showed an increase inthe rate of uptake and activation of macrophage cells. Cell cultureexperiments show the non-cytotoxicity and successful internalization ofthese chitosan-tannin nanoparticles. Accordingly, these novel compositenanoparticles can be used as targeted drug-delivery carriers. Thecomposites can also be used for a wide range of applications thatinvolve the efficient intracellular delivery of biological agents tomodulate the behavior of cells.

Tannin-chitosan composite nanoparticles can also be used as therapeuticbiomaterials, for example, to control pathogenic microbial colonizationof human epithelial cells. Because the nanoparticles themselves displayantimicrobial and antioxidant properties, contacting epithelial cellswith tannin-chitosan composite nanoparticles can kill, inhibit, orprevent the spread of a pathogenic microbes and microbial infections.Administration of the nanoparticles to a mammal can therefore treatand/or prevent human disease states such as diarrhea, resulting fromenterotoxigenic Escherichia coli (ETEC) and other pathogenic microbecolonization of the intestinal epithelial cells, and urinary tractinfections (UTI) resulting from adhesion of P-fimbriated uropathogenicbacteria to uroepithelial cells.

Additionally, tannin-chitosan composite nanoparticles can be used infeed additives, for example, to reduce shedding of E. coli O157:H7 incattle, and as a replacement for antibiotics in animal feeds. Thenanoparticles can also be used to replace herbicides, fungicides, and/orpesticides to control in-the-field and post harvest damage cause byplant pathogens.

Tissue Engineering.

The present generation of tissue engineering (TE) research is based onthe seeding of cells onto porous biodegradable polymer matrixes. Aprimary factor for successful seeding is the availability of goodbiomaterials to serve as the temporary matrix. Recently, chitosan andits derivatives have been reported as attractive candidates forscaffolding materials because they degrade as the new tissues areformed, eventually without inflammatory reactions or toxic degradation.In TE applications, the cationic nature of chitosan is primarilyresponsible for electrostatic interactions with anionicglycosylaminoglycans, proteoglycans, and other negatively chargedmolecules.

Tannin complexation to chitosan promotes a surface modification onchitosan films that increases the porosity of its 2D scaffolds, as shownby SEM micrographs. Tannins have also been found to improve the physicalproperties of chitosan biofilms, such as tensile strength, swellingdegree, and thermal stability, as shown by mechanical analysis and DSCcalorimetry. The chitosan-tannin composite polymer can be used tocontrol the morphology and function of cells, and therefore can be usedin tissue engineering, dermal drug delivery, and wound healingapplications. The chitosan-tannin composites can also be chemicallymodified for TE applications. For example, the composites can bemodified by grafting particular sugars to a tannin backbone. Certaincells can distinctively recognize the specific sugars, thus providingthe specific recognition to antigen presenting cells such as B-cells,dendritic cells, and macrophages.

Wound Healing.

Chitosan-tannin complexes (composites) also have valuable properties forwound healing applications because they exhibit enhanced bacteriostaticactivity with respect to pure chitosan. An increase in chitosanantimicrobial activity is observed in the chitosan-tannin complexes,which bind to the negatively charged bacterial surface to disturb thecell membrane. These properties can be applied to use of the complexesfor dermal patches, such as biofilms, for example, to promote ulcer andburn healing. The chitosan-tannin composites can also be used ashemostatic agents in wound dressings.

The chitosan-tannin complexes can be used in a variety of otherbiomedical applications. As a result of the biocompatible properties,such as good blood compatibility and cell growth efficiency,chitosan-tannin composites can be used in cardio-vascular applications.The permeability of chitosan-tannin composite membranes can becontrolled through plasma-treatment. Such composite membranes cantherefore be used in dialysis.

The preparation of wound dressings is described in U.S. Pat. No.7,482,503 (Gregory et al.). Wound dressings can be prepared according tosuch methods using the chitosan-tannin composite material describedherein in place of the chitosan biomaterial described therein.Additionally, the chitosan-tannin composites can be used as coatings formedical devices, such as stents or catheters, to prevent detrimentalbiofilm formation or bacteremia in patients.

Liposomes.

The chitosan-tannin composite material can be used to coat othercompositions, for example, using electrostatic deposition methods.Chitosan-tannin composite material can be electrostatic deposited ontoliposomes to increase their stability and provide enhanced delivery tospecific cells or tissues. Coated liposomes can be used as targetedrelease-on-demand carrier systems for both water- and oil-solublefunctional compounds such as drugs, antimicrobials, flavors,antioxidants, and other bioactive ingredients. The deposition ofchitosan-tannin composites improves the stability of liposomes, providesa more biodegradable and biocompatible coated liposome than currentsynthetic stabilization techniques, and adds antioxidant andantimicrobial functionality to the liposomes.

Liposomes coated with chitosan-tannin composite materials can providecontrast agents for use with diagnostic and therapeutic ultrasoundprocedures. Contrast agents such as Optison™ Perflutren Protein-Type AMicrospheres Injectable Suspension and Defininty® perflutrenencapsulated lipid microspheres are currently injected into systemicblood flow. Tannin-chitosan composites can be incorporated into suchliposomes to improve contrast features, assist in delivery oftherapeutic agents such as drugs or genes, or they can act alone astherapeutic agents.

Nutraceutical or Supplement Formulations.

The invention also provides formulations that include a tannin-chitosancomposite matrix described herein for use as a dietary supplement.Supplement compositions can be formulated as capsules, tablets, powders,solutions, gels, bar, suspensions, creams, and the like. These dietarysupplements, for example, in powder or solution form, can be added tonutraceuticals, foods and/or beverages to form pharmaceutical,functional nutraceutical, food, and/or beverage products. Thechitosan-tannin composite material can form a nanoparticle thatencapsulates additives such as vitamins or nutrients, or the compositematerial can be combined with an additive in a capsule, tablet, powder,solution, gel, bar, suspension, cream, or the like.

Dietary supplements may be formulated as powders, for example, formixing with consumable liquids such as milk, juice, water or consumablegels or syrups for mixing into other dietary liquids or foods. Dietarysupplements can be formulated with other foods or liquids to providepre-measured supplemental foods, such as single serving bars. Typicalfood products that can incorporate a tannin-chitosan composite matrixinclude dairy foods such as yogurt, cereals, breads, snack foodproducts, fruit juices, sports drinks and soft drinks Flavorings,binders, protein, complex carbohydrates, vitamins, minerals and the likecan be added as desired. Dietary supplements can be formulated bystandard techniques known to those of skill in the art, such as thetechniques described in U.S. Pat. No. 7,767,235 (Shrikhande et al.),which is incorporated herein by reference.

Examples of drugs, vitamins and nutrients that can be incorporated intoformulations include lipids such as fatty acids, including omega-3 andomega-6 fatty acids, fat soluble vitamins (e.g., vitamin A, D, E, and/orK), water soluble vitamins (e.g., vitamin C, thiamine, riboflavin,niacin, pantothenic acid, vitamin B6, folate, vitamin B12,), antibiotics(e.g., amoxicillin, ampicillin, clindamycin, doxycycline, erythromycin,metronidazole, penicillin, tetracycline, vancomycin, and the like),probiotics (e.g., lactic acid bacteria, bifidobacteria, and the like),micronutrients such as β-carotene and/or ascorbic acid, proteins, andpeptides. In some embodiments, monomeric tannins and/or othernutritional supplements can be incorporated into a chitosan-tannincomposite matrix, or they can be included in a composition that includesa chitosan-tannin composite matrix.

Analysis of Tannins, Chitosan, and Combination Products.

A variety of methods can be used to analyze and evaluate tannins,chitosans, and their composite products. These techniques include MatrixAssisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry(MALDI-TOF MS), American Standards for Testing Materials (ASTM)measurements of tensile strength and swelling properties, scanningelectron microscopy (SEM) to characterize surface morphology,differential scanning calorimetry (DSC) for thermal characterization ofthe composite biomaterial, and United States Pharmacopeia (USP) methodsto study drug release.

MALDI-TOF MS is especially suited to analyze the tannin-chitosancomposite materials because the technique can detect intact molecularions with high molar masses (e.g., >100,000 Da). MALDI-TOF MS is alsouseful for characterizing polydispersed oligomers that exhibit largestructural heterogeneity, including tannins, chitosans, and theircomposites. Tensile strength and swelling properties of the compositebiomaterials were characterized ASTM measurements.

Analysis of the tannin-chitosan composites described herein indicatesthat the composite materials have improved stability, higher drugloading capacity, improved drug release properties, improved celluptake, greater porosity, improved tensile strength and thermalstability compared to compositions that include only chitosan, and thematerials are non-cytotoxic in vitro.

Pharmaceutical Formulations.

The chitosan-tannin composite materials can be formulated aspharmaceutical compositions and administered to a mammalian host, suchas a human patient, in a variety of forms adapted to the chosen route ofadministration, e.g., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

The chitosan-tannin composite materials may be systemicallyadministered, e.g., orally, in combination with a pharmaceuticallyacceptable vehicle such as an inert diluent or an assimilable ediblecarrier. They may be enclosed in hard or soft shell gelatin capsules,may be compressed into tablets, or may be incorporated directly with thefood of the patient's diet. For oral therapeutic administration, thechitosan-tannin composite materials may be combined with one or moreexcipients and used in the form of ingestible tablets, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. Suchcompositions and preparations typically contain at least 0.1% of thechitosan-tannin composite materials. The percentage of the compositionsand preparations may, of course, be varied and may conveniently bebetween about 2% to about 60% of the weight of a given unit dosage form.The amount of chitosan-tannin composite materials in suchtherapeutically useful compositions is such that an effective dosagelevel will be obtained.

Tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the chitosan-tannincomposite materials may be used as a sustained-release preparation forthe administration of bioactive agents, such as drugs or othertherapeutic agents.

The chitosan-tannin composite materials may also be administeredintravenously or intraperitoneally by infusion or injection. Solutionsof the active compound or its salts can be prepared in water, optionallymixed with a nontoxic surfactant. Dispersions can also be prepared inglycerol, liquid polyethylene glycols, triacetin, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations may contain a preservative, for example, to prevent thegrowth of certain microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the chitosan-tannin composite materials which are adapted forthe extemporaneous preparation of sterile injectable or infusiblesolutions or dispersions. The chitosan-tannin composite materials mayalso be coatings for liposomes that encapsulate bioactive agents. In allcases, the ultimate dosage form should be sterile, fluid and stableunder the conditions of manufacture and storage. The liquid carrier orvehicle can be a solvent or liquid dispersion medium comprising, forexample, water, ethanol, a polyol (for example, glycerol, propyleneglycol, liquid polyethylene glycols, and the like), vegetable oils,nontoxic glyceryl esters, and suitable mixtures thereof. The properfluidity can be maintained, for example, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. The prevention of the action of certain microorganisms canbe brought about by various additional antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid,thiomersal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, methods of preparation include vacuumdrying and freeze drying techniques, which yield a powder of thechitosan-tannin composite materials plus any additional desiredingredient present in the previously sterile-filtered solutions.

For topical administration, the chitosan-tannin composite materials maybe applied in pure form. However, it will generally be desirable toadminister them to the skin as compositions or formulations, e.g., incombination with a dermatologically acceptable carrier, which may be asolid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, dimethyl sulfoxide (DMSO), alcohols or glycolsor water-alcohol/glycol blends, in which the chitosan-tannin compositematerials can be dissolved or dispersed at effective levels, optionallywith the aid of non-toxic surfactants. Adjuvants such as fragrances andadditional antimicrobial agents can be added to optimize the propertiesfor a given use. The resultant liquid compositions can be applied fromabsorbent pads, used to impregnate bandages and other dressings, orsprayed onto the affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the chitosan-tannin composite materials to the skin are known tothe art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392),Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157),and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the chitosan-tannin composite materials can bedetermined by comparing their in vitro activity, and in vivo activity inanimal models. Methods for the extrapolation of effective dosages inmice, and other animals, to humans are known to the art; for example,see U.S. Pat. No. 4,938,949 (Borch, et al.). The amount of thechitosan-tannin composite materials required for use in treatment willvary not only with the particular composite form used but also with theroute of administration, the nature of the condition being treated andthe age and condition of the patient and will be ultimately at thediscretion of the attendant physician or clinician. For example, thechitosan-tannin composite materials may be conveniently administered inunit dosage form; for example, containing 5 to 1000 mg/m², 10 to 750mg/m², or 50 to 500 mg/m² of active ingredient per unit dosage form.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations.

The present invention provides therapeutic methods of treating variousconditions in a mammal, which involve administering to a mammal havingsuch a condition an effective amount of a chitosan-tannin composite ofthe invention. A mammal includes a primate, human, rodent, canine,feline, bovine, ovine, equine, swine, caprine, bovine and the like. Inother embodiments, the chitosan-tannin composite materials can be usedto treat various conditions in plants, such as bacterial or fungalinfections.

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest other ways in whichthe invention could be practiced. It should be understood that numerousvariations and modifications may be made while remaining within thescope of the invention.

EXAMPLES Example 1 Tannin-Chitosan Composites: Preparation, Data, andApplications

Pharmaceutical grade chitosan (deacetylation degree of 92% calculated by¹H NMR; mean molecular weight of 185 kDa calculated by specificviscosimetry) derived from shrimp shells was provided by the PolymersResearch Laboratory (POLIUNA) of Costa Rica. The degree of deacetylationand mean molecular weight distribution can be controlled in theproduction of tannin-chitosan composites to provide chitosan with higheror lower degrees of deacetylation, and/or higher or lower mean molecularweights. Chitosan-tannin composites that included hydrolyzable tannins(HT) and proanthocyanidins (PA) were separately prepared and analyzed.

Proanthocyanidins (PA).

Polyphenolics were isolated by liquid chromatography and subjected toMALDI-TOF MS using trans-3-indoleacrylic acid as the matrix. Spectralanalysis provided the degree of polymerization, monomeric substitution,and the nature of intermolecular bonds (Reed et al. 2005).

The cranberry polyflavan-3-ols had variation in interflavan bonds(A-type and B-type) and contained polyflavan-3-ols linked toanthocyanins through a CH₃—CH bridge (see Scheme 1 above) (Krueger etal. 2004). Polygalloyl-polyflavan-3-ols in grape seed extract had largevariation in the degree of galloyl substitution (Krueger et al. 2000).Sorghum polyflavans had structural heterogeneity in glycosylation andhydroxylation (Krueger et al. 2003).

Cranberry polyphenols were chosen as a representative for the class of‘proanthocyanidins’ in formulations of Tannin (PA)-Chitosanbioconjugates. Spray dried cranberry juice powder was reconstituted inH₂O and applied to a preparative LH-20 column equilibrated in water.Water, 50% aqueous ethanol (v/v), and ethanol were sequentially elutedthrough the column to remove non-phenolic cranberry constituents as wellas monomeric phenolic constituents (hydroxycinnamic acids, anthocyaninsand flavonols). Aqueous acetone (4:1; acetone: H₂O, v:v) was then passedthrough the column until it was white, to elute a mixture of cranberryPA. The aqueous acetone fraction was concentrated by vacuum to removethe acetone and the water was removed by freeze drying.

Other solid phase chromatographic resins (C18, C8, Waters Oasis resins,Amerlite resins, and the like) can also be used to enhance extraction ofparticular tannin fractions. Likewise, liquid/liquid (partition)chromatography can be used for extractions, as well as supercriticalfluid extraction techniques. Additional techniques that can be used forthe isolation and purification of various tannins are described in U.S.Pat. No. 7,122,574 (Romanczyk et al.), which is incorporated herein byreference.

The cranberry proanthocyanidin composition was characterized byMALDI-TOF MS, showing a degree of polymerization (DP) ranging from 4 to7, with at least one A-type interflavan bond present at for each degreeof polymerization. FIG. 1 illustrates a MALDI-TOF MS spectrum, inpositive reflectron mode, of the cranberry PA fraction. The sectionbetween m/z 1000 and 2500 has been amplified to identify the degree ofpolymerization, the nature of interflavan linkage and structuralcomposition of subunits of oligomeric PA. The degree of polymerization,monomeric substitution, and nature of intermolecular bonds are‘proanthocyanidin variables’ that can be controlled by choice of rawstarting material (plants, fruits, juices), extraction processes, andenrichments by liquid and solid phase chromatography.

Hydrolysable Tannins (HT).

Hydrolysable tannins were isolated from pomegranate peels. Thehydrolysable tannins identified included gallic acid and ellagic acidesters of core polyol moieties, such as sugars (see Scheme 4 above).Pomegranate hydrolysable tannins that correspond to previously describedstructures, such as punicalagin, were identified. Others found werehydrolysable tannins that correspond to oligomeric ellgitannins in whichtwo to five core glucose units are cross linked by dehydrodigalloyl andor valoneoyl units (Afaq et al. 2005 and Seeram et al. 2006). Theseresults demonstrate the degree of polymerization, intermolecular bonds,pattern of hydroxylation, and substitution with monosaccharides andgallic acid, occur with an identifiable amount of heterogeneity.

HT Isolation.

Pomegranate polyphenols were chosen as t representative for the class of‘Hydrolyzable Tannins’ for formulations of Tannin (HT)-Chitosanbioconjugates. Pomegranate peels (˜5 g) were added to liquid nitrogen ina blender and ground to a powder. The powder was extracted with aqueousacetone 80% v/v, filtered, and concentrated by vacuum. Concentratedpomegranate extract was applied to a C18 preparative column equilibratedwith water. Water was passed through the column to elute non-phenolicconstituents. To elute a pomegranate HT fraction enriched inpunicalagin, an aqueous methanol 50% (v/v) solution was then passedthrough the column until the resin became white.

MALDI-TOF MS and RP-HPLC analysis showed that the pomegranate HTfraction was composed mainly of punicalagin (˜95%). FIG. 2 illustrates aMALDI-TOF MS spectrum, in negative reflectron mode, of the pomegranatehydrolyzable tannin. The degree of polymerization, monomericsubstitution, and nature of intermolecular bonds are ‘hydrolyzabletannin variables’ that can be controlled by choice of raw startingmaterial (plants, fruits, juices), extraction processes and enrichmentsby liquid and solid phase chromatography.

Preparation of Tannin-Chitosan Composites (Bioconjugates).

A chitosan solution was prepared by dissolving chitosan powder in anacetic acid aqueous solution (0.5% v/v) at room temperature (˜23° C.).After the chitosan powder was fully dissolved, the solution was filteredand degassed by vacuum filtration (Step 1). FIG. 3 schematicallyillustrates the dissolving and degassing the chitosan solution. Thechitosan solution (0.5% w/v) was then mixed with eitherproanthocyanidins or hydrolyzable tannins (0.5-2.0% v/v), at differentmolar ratios. The solution was left to react, with stirring, for 1 hourat room temperature (Step 2). FIG. 4 further illustrates the preparationof the chitosan-tannin bioconjugates, according to an embodiment. Theconcentration of chitosan and tannins (PA and/or HT) were controlled inthe process of forming composite materials by adding different ratios ofthe respective components to provide the desired composition.

Preparation of Tannin-Chitosan Composite 2D Hydrogels (Films).

The tannin-chitosan composite solutions were cast onto glass plates andslowly spread to form even liquid films. The liquid films were thenevaporated at room temperature for 24 hours to form 2D biomembranes(hydrogels). The hydrogel films were subsequently washed repeatedly withdeionized water and were dried at room temperature for 24 hours (Step 3a) to provide the composite films. FIG. 5 illustrates the casting of thechitosan-tannin hydrogels, according to an embodiment.

Preparation of Tannin-Chitosan Composite Nanoparticles.

Chitosan-tannin nanoparticles were prepared by ionitropic gelation ofchitosan with tripolyphosphate (TPP) anions. TPP was dissolved in waterto a concentration of 1 mg/mL. Under magnetic stirring at roomtemperature, 2 mL of TPP solution were added to 5 mL of thetannin-chitosan composite solutions, as illustrated in FIG. 6. Themixture was stirred for 30 minutes followed by sonication (Step 3 b).Composite nanoparticles were filtered and freeze-dried to providefree-flowing nanoparticles.

Preparation of Tannin-Chitosan Composite 3-D Bio-foams.

Composite foams (diameter=12 mm, thickness=6 mm) were prepared bycasting/freeze-drying techniques (Step 3 c). One gram of a 2% w/wchitosan solution in water or 0.5% v/v in acetic acid was mixed withtannins aqueous solutions (0.5-2.5% v/v), as illustrated in FIG. 7. Theresulting mixture was poured into a cylindrical mold of adequate size,frozen at −20° C., and freeze-dried to eliminate the solvent, to providethe tannin-chitosan composite porous (3D) foam. The tannin-chitosancomposite bio-foams are physically similar to known chitosan foams,however the composite bio-foams possess significant additionalproperties. The tannin-chitosan composite bio-foams can be used, forexample, to provide improved wound and hemostatic (blood coagulation)dressings because the hemostatic effect of chitosan is increased by theimmunostatic properties of tannin component.

Preparation of Tannin-Chitosan Composite Biogels.

A chitosan (1% w/v)-tannin (2% v/v) bioconjugate solution was frozen at−20° C. and freeze-dried to eliminate the solvent, leaving a pigmentedpowder material. One to two grams of the lyophilized chitosan-tanninbioconjugate was dissolved in 100 mL of distilled or deionized water andstirred vigorously while the pH was increased dropwise with aconcentrated solution of NaOH 6N, as illustrated in FIG. 8. Once thesolution reached an adequate pH value (˜7.2), a bio-gel spontaneouslyformed and the viscosity of the dispersion significantly increased (Step3 d).

Physical and Chemical Characterization of Tannin-Chitosan Composites.

A Panasonic Hi-Star II X-ray diffractometer (XRD) was used toinvestigate the crystal structure of the synthesized tannin-chitosancomposites. The X-ray source was Ni-filtered Cu—Kα radiation (40 kV, 30mA). A Perkin-Elmer DSC 7 differential scanning calorimeter (DSC) wasused to evaluate the thermal properties of the chitosan and thechitosan-tannin bioconjugates (about 3 mg) under N₂ atmosphere at aheating rate of 20 K/minute from 50° C. to 400° C.

X-ray diffraction patterns of tannin-chitosan composites were comparedwith chitosan. FIG. 9 illustrates X-ray diffraction patterns of chitosanand tannin-chitosan composites. Arrows indicate significant changesgenerated by the tannin component on the crystal arrangement of thebiopolymer. Results showed an increase on the crystalline structure ofboth HT and PA-chitosan composites, as indicated by the increase of theintensity of the crystalline segments at 2θ=8° and 2θ=12°, respectively.Each peak represents a modification of the chemical structure of themain chitosan polymeric backbone that is related to either stronghydrogen bonding interaction or covalent bonding with the HT and PATannin.

Thermal analysis of the tannin-chitosan composites by DSC showed newthermic transitions appearing for both PA tannin-chitosan and HTtannin-chitosan composites. FIG. 10 illustrates DSC thermograms ofchitosan (top dashed line) and tannin-chitosan composites (bottom solidline). FIG. 10A illustrates HT-derived composites and FIG. 10Billustrates PAC-derived composites. Arrows indicate significant changeson the thermal behavior of the composite material. HT-chitosan compositeshowed an increase on the enthalpy associated with an endothermictransition at 50° C. and also a new endothermic transition appears at130° C. Meanwhile, the PAC-chitosan composite showed a higher structuralcomplexity that increases the rigidity of the biomolecule as shown bythe disappearance of previously observed endothermic transitions ofchitosan at 50° C. and 320° C. Both DSC thermograms for chitosan-tanninbioconjugates indicated a change in the main polymeric network due tothe addition of the tannins, directly affecting the thermal stability ofthe macromolecule.

X-ray diffraction and DSC thermograms provided clear evidence of theformation of a new stable composite biomaterial. These analytic toolscan be used to aid further optimization of processing and formulation ofthe new tannin-chitosan composites to meet desired physical chemicalproperties.

Characterization of Chitosan-Tannin Hydrogels (Films).

The mechanical properties of the tannin-chitosan composite hydrogelswere evaluated by comparing tensile strength and swelling behavior.Swelling is the first step in the physical degradation of hydrogels.Rapid swelling promotes a rapid and uncontrolled release of activecompounds (e.g., drugs and/or pesticides) from a hydrogel matrix.Glutaraldehyde is commonly added as a cross linking agent in theproduction of chitosan hydrogels to slow the rate of swelling. Adisadvantage of using glutaraldehyde in a hydrogel formulation is areduction in the tensile strength of the hydrogel.

Chitosan and PA- and HT-chitosan composite hydrogels were cast accordingto the previous described methodologies. In addition, ‘crosslinkedhydrogels’ were formulated by first immersing pre-cast chitosan ortannin-chitosan composite hydrogels in a glutaraldehyde solution (0.10%v/v) for 30 minutes, followed by exhaustive washing with deionizedwater, followed by drying at 80° C. for 2 hours.

Mechanical Properties of Chitosan-Tannin Composite Hydrogels.

Tensile strength measurements were performed on a tensile testingmachine (model TEST 108 from GT Test, France, equipped with Test Winner920 software), with a crosshead speed of 10 mm/minute and a 2 kN staticload cell. The hydrogels were cut into standard tensile samples from adumbbell-shaped knife (H3 type) with a dimension 17 mm×4 mm×0.08 mm(length×width×thickness). At least five samples of each type of hydrogelwere tested after a suitable storage period (3 and 20 weeks) at 50±3% RHand 23±2° C. in a humidity chamber (CIAT, France). The maximal tensilestress (TS) was calculated by dividing the maximum load for breakingfilm by cross-sectional area.

Both the PA-Chitosan composite hydrogel (no glutaraldehyde) and theHT-Chitosan composite hydrogel (no glutaraldehyde) showed higher tensilestrength than the chitosan hydrogel alone (no glutaraldehyde). FIG. 11illustrates the mechanical behavior of chitosan-tannin hydrogelscompared to a chitosan hydrogel. Data is shown as [mean±SD; n=5]. Theaddition of a crosslinking agent (glutaraldehyde) reduced tensilestrength of all hydrogels relative to the non-crosslinked hydrogels.There was no observed difference in tensile strength between crosslinkedchitosan hydrogels and crosslinked tannin-chitosan composite hydrogels.

Evaluation of Swelling Behavior.

The degree of swelling of chitosan and tannin-chitosan hydrogels wasevaluated by gravimetric methods. Each dry hydrogel was first weighed onan analytic balance (Wd). After weighing, hydrogels were submerged indistilled water for 60 minutes at room temperature. Hydrogels were thenremoved from the water and weighed (Ws) at 5, 10, 20, 30, 40, 50, and 60minutes. Prior to being weighed on a high precision balance, each filmsample was quickly taken out from the water bath and blotted with tissuepaper to remove excess water. After weighing, the hydrogels werereturned to the water. The degree of swelling (%) of each film samplewas then calculated according to the following equation:

$\begin{matrix}{{{Degree}\mspace{14mu}{of}\mspace{14mu}{swelling}\mspace{14mu}(\%)} = {\frac{W_{s} - W_{d}}{W_{d}} \times 100.}} & (2)\end{matrix}$

The results indicate that both PA and HT Tannin-Chitosan compositehydrogels, with no glutaraldehyde crosslinking agent, showed lower ratesof swelling and lower total degrees of swelling compared to chitosanhydrogels with no crosslinking FIG. 12 illustrates the degree ofswelling for the chitosan-tannin hydrogels as a function of time.

These results indicate that tannins act as a strong crosslinking agentor the equivalent thereof, reducing the swelling of the compositematerials while improving the tensile strength of the hydrogel. Theproperties provided by tannins were superior to those of aglutaraldehyde crosslinking agent in chitosan hydrogels. Tannins aretherefore suitable, reliable, and biocompatible ‘green alternatives’ toglutaraldehyde for formulating hydrogels for packaging, patching, andsurgical biomaterials.

Characterization of Chitosan-Tannin Nanoparticles.

Size and Zeta Potential.

Size determinations and electrophoretic mobility measurements werecarried out with a Z-Meter Zetasizer 2000 from Malvern Instruments andalso with a Z-Meter System 3.0 from Z-Meter USA equipped with microscopemodel DR from Carl Zeiss and an electrophoresis cell GT2 type. For sizeand electrophoretic mobility measurements, the samples were obtained asstated above and afterwards diluted to 1:10 with deionized water. Fivesamples were prepared for each tannin-chitosan composite ratio. Theerror was the highest standard deviation for the five samples. Forillustrative purposes, approximate ζ-potential values were calculatedstarting from the Smoluchowski's equation, and using the followingvalues: ε_(o)=8.9×10⁻¹² Fm⁻¹ and ε_(r)=79.

The results shown in Table 1-1 indicate that both particle size andζ-potential of the chitosan nanoparticles (ChNp) increased as a functionof the biopolymer concentration, showing a critical concentration around0.15% w/v. After this value ChNp appears to suffer coalescence andaggregate, as indicated by the drastic increase on particle size after0.10% w/v. According to this observation, chitosan-PA tanninnanoparticles (ChPANp) and chitosan-HT tannin nanoparticles (ChHTNp)where formulated holding the concentration of chitosan solutions (0.10%w/v) constant and varying the amount of tannin (5%, 10%, and 20%).

TABLE 1-1 Variation in size and ζ-potential for different chitosan andchitosan-tannin nanoparticle formulations. Results are shown as [mean ±SD, n = 5]. ζ-Potential Size Sample ID Study Size (nm) (mV) (nm)* ChNp0.05% Effect of chitosan 130 ± 1 19.6 ± 0.9 152 ± 3 ChNp 0.10%concentration 149 ± 1 20.9 ± 1.9 ChNp 0.20%  426 ± 28 32.2 ± 1.6 ChHTNp5% Effect of HT 165 ± 4 24.4 ± 0.5 165 ± 2 ChHTNp 10% concentration 167± 3 22.8 ± 1.5 ChHTNp 20% (chitosan 0.1% w/v) 165 ± 4 22.6 ± 2.5 ChPANp5% Effect of PA 298 ± 7 23.2 ± 3.1 298 ± 3 ChPANp 10% concentration 296± 2 21.5 ± 1.0 ChPANp 20% (chitosan 0.1% w/v) 292 ± 4 20.6 ± 0.9 *Sizeof nanoparticles after being reconstituted in water followinglyophilization.

ChPANp and ChHTNp size and ζ-potential were not affected by the tanninconcentration. This observation indicates an intermolecular interactionbetween the components and not simply a physical adsorption on thesurface structure of the biopolymer that increased the size and changedthe ζ-potential of the nanoparticles. Nanoparticle size afterlyophilization and reconstitution in water were not significantlydifferent from original particle size, indicating that thesebiomaterials can be stored and marketed in powder form.

ζ-Potential is an important parameter for stability in aqueousnanosuspensions. For a physically stable nanosuspension, solelystabilized by electrostatic repulsion, a ζ-potential of ±30 mV isrequired as a minimum. All data indicated that tannin-chitosan compositenanoparticles are stable and can be dried by lyophilization withoutchanging the average particle size.

Surface and Morphology Analysis.

The nanostructure of CNp, CPANp and CHTNp were examined on a JEOLJSM-5200 transmission electron microscope (TEM) with a tilt angle of 30°and by atomic force microscopy (AFM) on an Asylum Research AFM equippedwith 3D imaging software.

Results of the surface and morphology analysis confirmed the presence ofnanometric particles for both chitosan and tannin-chitosan compositesystems. FIG. 13 illustrates scanning electron microscopy (SEM)micrographs for chitosan and chitosan-tannin nanoparticles: 13A: ChNp;13B: ChPANp; and 13C: ChHTNp. The scale bar indicates 200 nm. These SEMmicrographs clearly show the appreciable increase in the crystallinestructure of the tannin-chitosan composites (FIGS. 13B and 13C) comparedto the amorphous chitosan nanoparticles (FIG. 13A).

Chitosan nanoparticles are significantly smaller than thetannin-chitosan nanoparticles. Additionally, TEM micrographs showagglomeration of chitosan nanoparticles, indicated by white, cloudyareas on the TEM micrographs (data not shown), indicating instability ofthe chitosan nanoparticles. Tannin-chitosan nanoparticles showedconsistent shapes and homogeneous size distributions that confirm theirsuperior physical performance and stability.

Analysis of atomic force microscopy (AFM) micrographs confirmed that theparticle size and surface characteristics of the tannin-chitosancomposites are similar when compared with the standard chitosannanoparticles. AFM micrographs show that when chitosan is complexed totannins, there is a change in both shape and surface/distributionbehavior compared to chitosan nanoparticles. Chitosan-TPP nanoparticlesare typically spherical and lack agglomeration (due to surface chargerepulsions), whereas chitosan-tannin-TPP nanoparticles showedcrystalline forms that trend to agglomerate via hydrogen bonding,indicating an increase in surface polarity due to the presence of thetannins The increase in crystalline structure of the tannin-chitosancomposites provides several advantages, including improved performanceof the tannin-chitosan composites as carriers for therapeutic agent orpesticide applications.

Bacteriostatic and Fungistatic Action of Tannin-Chitosan CompositeNanoparticles.

Botrytis cinerea, also referred to as grey mold or botrytis bunch rot,has an economic impact on soft fruits such as strawberries and grapes.Fusarium oxysporum causes Fusarium wilt disease in more than a hundredspecies of plants, resulting in leaf wilting, yellowing, and eventuallyplant death. Erwinia carotovora is a bacteria that infects a variety ofvegetables, such as carrots, potatoes, cucumbers, onions, tomatoes, andlettuce, in fields or in storage, causing plant tissues to become softand watery, which eventually deteriorate and become foul-smelling.Xanthomonas is a bacteria that affects many types of commercial plants,such as citrus, beans, grapes, cotton, and rice, causing lesions on theleaves, fruit, and stems, as well as twig dieback.

Chitosan-tannin composite nanoparticles were tested for their ability toprevent growth of agricultural pathogenic stains of fungi (Bothytiscines and Fusarium oxysporum) and bacteria (Erwinia carotovora andXanthomonas spp.). Results indicate that both proanthocyanidin-chitosannanoparticles (CPACNp) and hydrolysable tannin-chitosan nanoparticles(CHTNp) increase inhibition of all pathogens 2-3 fold compared tochitosan nanoparticles (CNp) alone. FIG. 14 illustrates theantimicrobial susceptibility test of these tannin-chitosan conjugateagainst common Costa Rican agricultural pathogens. Data is shown as[mean±SD; n=3].

Characterization of Chitosan-Tannin Composite 3-D Bio-Foams: Surface andMorphology Analysis.

Scanning electron microscopy (SEM) micrographs at 7 Kv showed excellent3-D structures of the tannin-chitosan composites 3-D biofoams,confirming stability of composite material.

Advantages of Tannin-Chitosan Composite Biomaterials Compared toChitosan Biomaterials.

The tannin-chitosan composite biomaterials provide significantlyimproved properties for a variety of applications, compared to chitosanbiomaterials. The tannin-chitosan composite biomaterials can be preparedas nanoparticles, hydrogels such as bio-films, 3D-biofoams, or biogels.Each of these forms of biomaterials can be used for various targetapplications, and each the composite biomaterial has significantadvantages over chitosan biomaterials, as summarized in Table 1-2 below.

TABLE 1-2 Improvements of Tannin-Chitosan Composite BiomaterialsCompared to Chitosan Biomaterials. Improvement using Biomaterial Targetapplication chitosan-tannin composites Nanoparticles Controlled drugrelease Tannin's crosslinking effect increases Antimicrobial propertiescontrolled released Biodegradability Tannins impart bacteriostatic andfungistatic activity to nanoparticles Tannins decrease the rate ofbiodegradation of the nanoparticles, an important property to promotesustained delivery Hydrogels Controlled drug release Tannin'scrosslinking effect increases (bio-films) Mechanical propertiescontrolled released; can be a replacement Antimicrobial properties forglutaraldehyde Biodegradability Chitosan-tannin hydrogels showed anWound healing increase in tensile strength compared to chitosan; thecomposites are therefore suitable for dressing, patch, and packagingbiomaterial applications Tannin's increase the nanoparticles'interaction with bacteria and fungi Tannins decrease the rate ofbiodegradation of the biofilms, an important property to promotesustained delivery Tannin's immunostatic properties can increase abiofilm's performance as wound healing promoters 3D-BiofoamsAntimicrobial properties Tannins can increase bio-foam interactionBiocompatibility with bacteria and fungi, thereby becoming Wound healingand more active and suitable as dermal coagulation agent patches orbandages Cell growth and tissue Tannin's immunostatic properties canengineering increase biofoam performance as wound healing andcoagulation promoters Chitosan-tannin bioconjugate biocompatibility,antimicrobial properties, and porosity made them an advantageousscaffolding material for tissue engineering Biogels BiocompatibilityTannins are non-immunogenic and are Additive biomaterial suitabledietary supplements Chitosan-tannin biogels can be readily used asadditive biomaterials in various formulations (e.g., food, adhesives,pharmaceuticals, biomedical applications) due to their improvedmucoadhesion and viscosity, as well as their antimicrobial andimmunostatic properties

Supporting information can be found in the following documents, whichare incorporated herein by reference.

-   Afaq F., Saleem M., Krueger C. G., Reed J. D., Mukhtar H. 2005.    Anthocyanin- and hydrolyzable tannin-rich pomegranate fruit extract    modulates MAPK and NF-kappaB pathways and inhibits skin    tumorigenesis in CD-1 mice. Int. J. Cancer 113 (3): 423-433.-   Howell A. B., Reed J. D., Krueger C. G., Winterbottom R.,    Cunningham D. G., Leahy M. 2005. A-type cranberry proanthocyanidins    and uropathogenic bacterial anti-adhesion activity. Phytochem.    66(18):2281-2291.-   Krueger, C. G., M. M. Vestling and J. D. Reed. Matrix-assisted laser    desorption/Ionization time-of-flight mass spectrometry of    anthocyanin-polyflavan-3-ol polymers in cranberry fruit [Vaccinium    macrocarpon, Ait.] and spray dried cranberry juice. ACS Symposium,    Uncovering the Mysteries of Red Wine Pigments. Vol. 886: pp.    232-246. 2004.-   Krueger, C. G.; Vestling, M. M.; Reed, J. D. Matrix-assisted laser    desorption/ionization time-of-flight mass spectrometry of    heteropolyflavan-3-ols and glucosylated heteropolyflavans in sorghum    [Sorghum bicolor (L.) Moench)]. J. Agric. Food Chem. 2003. 53,    538-543.-   Krueger, C. G., N. C. Dopke, P. M. Treichel, J. Folts, and J. D.    Reed. 2000. Matrix-assisted laser desorption/ionization    time-of-flight mass spectrometry of polygalloyl polyflavan-3-ols in    grape seed extract. J Agric. Food Chem. 47: 3693-3701.-   Ravi Kumar, M. N. V, Muzzarelli, R. A. A., Muzzarelli, C.,    Sashiwa, H. and Domb, A. J. 2004. Chitosan chemistry and    pharmaceutical perspectives. Chemical Reviews. 104:6017-6084.-   Reed J. D., Krueger C. G., Vestling M. M. 2005. MALDI-TOF Mass    spectrometry of oligomeric food polyphenols. Phytochem. 66(18):    2248-2263.-   Rinaudo, M. 2006. Chitin and chitosan: Properties and applications.    Progress in Polymer Science. 31:603-632.-   Seeram, N. P., Zhang Y., Reed J. D., Krueger C. G., Yaya J. 2006.    Pomegranate Phytochemicals. In Pomegranates (Medicinal and Aromatic    Plants—Industrial Profiles). Editors: Seeram, N. P., Schulman, R. N.    and Heber, D.

Example 2 Tannin-Chitosan Composites: Secondary Liposomes Stabilized byElectrostatic Deposition of Tannin-Chitosan Composites

Liposomes are spherical bilayer vesicles formed by dispersions ofcertain polar lipids in aqueous solvents. Liposomes have attractedattention in the food and agricultural industries because of theirability to act as targeted release-on-demand carrier systems for bothwater- and oil-soluble functional compounds, such as antimicrobials,flavors, antioxidants, and bioactive ingredients (Benech et al. 2002;Were et al. 2003). Encapsulation of functional components in liposomeshas been shown to increase their stability and maintain their activityin environments that typically lead to rapid degradation.

One major limitations of liposomes is that they have a tendency to leakand lose encapsulated components over time (Taylor et al. 2005). Gradualcoalescence of liposomes may also occur. Coalescence becomes morepronounced in low pH environments where surface charges are reduced(Gregoriadis 1973; Gregoriadis 1993). Adsorption of a second layer ontothe liposome, such as a tannin chitosan composite material as describedherein, can improve the stability of liposomes and provide aninexpensive means to tailor the surface properties of liposomes.Additional layers (e.g., a third, fourth, and/or fifth layer) can beadded to modify controlled release properties. These layers may alterbetween chitosan and tannin (PAC or HT) or they may be tannin-chitosancomposites themselves, to provide different composition properties.

The electrostatic deposition of tannin-chitosan composites ontoliposomes is an improvement over current compositions becausetannin-chitosan composites are more biodegradable and biocompatible thancurrently used synthetic agents, such as polyethylene glycol and otherpolymers, in stabilization of liposomes. Tannin-chitosan composites alsoprovide improved compositions because they impart valuable properties,such as anti-microbial and antioxidant properties, to the liposome inaddition to improving the liposome stability. Tannin-chitosan compositecoated liposomes can be used, for example, as diagnostic or therapeuticagents in contrast enhanced ultrasound.

Preparation of Secondary Liposomes Coated by Tannin-Chitosan Composites.

Secondary liposomes were prepared from concentrated soy lecithindispersions according to procedures previously described(Madrigal-Carballo et al. 2007). Briefly, soy lecithin dispersions (250g/L) were prepared by the slow-swelling-under-shear method in an aqueousmedium (10⁻⁵ M NaCl). Chitosan-tannin solutions ranging 0 to 10% v/vwere produced from an aqueous solution of chitosan (0.10% w/v in aceticacid 0.5% v/v) complexed with cranberry proanthocyanidins (PAC) orpomegranate hydrolysable tannins (HT). Liposome/chitosan-tannindispersions were prepared by diluting the previously preparedconcentrated soy lecithin dispersions with the chitosan-tannin complexessolutions until a final concentration of 3 g/L was reached.

Size determinations and electrophoretic mobility measurements werecarried out with a Z-Meter Zetasizer 2000 from Malvern Instruments andalso with a Z-Meter System 3.0 from Z-Meter USA equipped with microscopemodel DR from Carl Zeiss and an electrophoresis cell GT2 type. All theζ-potential values were approximated by the Smoluchowski's equation.

Influence of Chitosan-Tannin Concentration on Properties of CoatedLiposomes.

The interaction between chitosan-tannin complexes and liposomes (dapproximately 200 nm) was monitored by measuring the electrical chargeand mean diameter of the particles in mixed liposome suspensions at pH 6(FIGS. 15 and 16). FIG. 15 illustrates the influence of addition ofchitosan-tannin (0 to 0.5, w/v %) to liposomes (lecithin 0.4% w/v, 200nm) on the ζ-potential values of the vesicles. ζ-Potential measurementsindicated that the uncoated liposomes were highly anionic (−38 mV),whereas the chitosan-tannin molecules were highly cationic (+82 mV). Thesurface charge of the particles in liposome suspensions (lecithin 0.4%w/v) increased from −38 mV to +30 mV with addition of chitosan-tannins(0% to 10% v/v).

The net charge on the particles was zero after addition of approximately3% v/v chitosan-tannin complexes, indicating that charge neutralizationoccurred at this liposome-to-complex composition. The observed changesin surface charge suggest that chitosan-tannin complexes adsorbed to thesurfaces of liposomes until the liposomal membrane was fully coveredwith the complex molecules thereby preventing further adsorption.

The mean diameter of the particles in the suspensions was highlydependent on the concentration of chitosan-tannin added to the system(FIG. 16). The particle diameter increased from around 200 nm in theabsence of the complex to well above 4000 nm in the presence of lowamounts of added chitosan-tannin complex (3% v/v), indicating theformation of large aggregated structures. These aggregates eventuallyphase separated and formed a precipitate at the bottom of the test tube.The range of chitosan-tannin concentrations where large aggregates wasformed corresponded to the surface charge of the particles changing fromapproximately −20 mV to +20 mV, which suggested that this type ofaggregation was caused by charge neutralization or possibly bridging(FIG. 15). At chitosan-tannin concentrations above 3% v/v, the particlediameter decreased to below about 500 nm to reach a minimum value at achitosan-tannin concentration of 5% v/v. Further addition of chitosanled to steady increases in particle diameter. For example, the particlediameter was approximately 700 nm after addition of complex at 10% v/v.

Adsorption of Chitosan-Tannin onto Liposome Surfaces.

Electrostatic interactions between charged particles and chargedbiopolymers have been extensively studied, but there are few detailedinvestigations of the interaction of shell-structured liposomes withoppositely charged biopolymers. To this purpose, chitosan-tannins wereadded to liposomes under controlled conditions (rate of stirring,solution composition, temperature). The limitation of using chargedbiopolymers with too low or too high a molecular weight to form a stablebiopolymer coating around liquid or solid particles has been previouslyexplained in terms of the charge-mosaic theory, and a detailedexplanation of the theory is described by Henriksen et al. 1997.

The results of the studies described herein indicate that liposomesinteracted strongly with chitosan-tannins via electrostatic interactionsto form a range of structures, depending on the ratio of chitosan-tanninto liposomes. Addition of a charged biopolymer to a dispersion ofoppositely charged particles causes the electrical charge on theparticles to change from either positive to negative, if particles arecationic and an anionic biopolymer is added, or vice versa if particlesare anionic and a cationic biopolymer is added (Guzey and McClements2007; Hong and McClements 2007; Pallandre et al. 2007). Stablechitosan-tannin coated liposomes were formed within only a narrowconcentration range (c_(min)<c<c_(max)). Below and above this optimalrange liposomes aggregated and eventually phase separated from solution(FIG. 16). The minimal concentration required to form stable secondaryliposomes can be estimated from the change in ζ-potential with additionof chitosan-tannin complexes.

Stability of Liposomes Upon Addition of Chitosan.

Addition of chitosan-tannins to liposomes below and above the saturationconcentration led to destabilization of the liposomal dispersions (FIGS.15 and 16). The different structures that are observed when theconcentration of chitosan-tannin was either too low or too high suggestthat two different mechanisms can occur. At insufficient complexconcentrations, liposomes completely broke down to rapidly (withinminutes) form sediment at the bottom of the test tube. This could bebecause the anionic phospholipid molecules may have bound to thecationic chitosan-tannin molecules to form coacervates as opposed to thecationic chitosan-tannin molecules wrapping themselves around thesurfaces of the liposome particles.

In the presence of excess concentration of complex, the mechanism may besimilar to that found in emulsions where depletion flocculation mayoccur (Guzey and McClements 2006). The excess concentration of polymermay create an osmotic pressure gradient due to the exclusion of polymermolecules from the immediate vicinity of the particle surfaces, whichgenerates at attractive force that increases with increasing complexconcentration. At sufficiently high concentrations of non-adsorbedcomplex, this depletion attraction is sufficient to promote particleaggregation (McClements 2005). The model described herein for theobserved instability phenomena upon addition of chitosan-tannincomplexes to liposomes is shown in FIG. 17.

Supporting information can be found in the following documents, whichare incorporated herein by reference.

-   1. Benech et al. 2002. Inhibition of Listeria innocua in Cheddar    cheese by addition of nisin Z in liposomes or by in situ production    in mixed culture. Appl. Environ. Microbiol. 68:3683-90.-   2. Gregoriadis G. 1973. Drug entrapment in liposomes. FEBS Lett.    36:292-6.-   3. Gregoriadis G. 1993. Liposome technology. 2nd ed. Boca Raton,    Fla.: CRC Press.-   4. Were et al. 2003. Size, stability, and entrapment efficiency of    phospholipid nanocapsules containing polypeptide antimicrobials. J.    Agric. Food Chem. 51:8073-9.-   5. Taylor et al. 2005. Liposomal nanocapsules in food science and    agriculture. Crit. Rev. Food Sci. Nutr. 45:1-19.-   6. Guzey D, McClements D J. 2006. Formation, stability and    properties of multilayer emulsions for application in the food    industry. Adv. Colloid Interface Sci. 130:227-48.-   7. Guzey D, McClements D J. 2007. Impact of electrostatic    interactions on formation and stability of emulsions containing oil    droplets coated by beta-lactoglobulin-pectin complexes. J. Agric.    Food Chem. 55(2):475-85.-   8. Henriksen et al. 1997. Interactions between liposomes and    chitosan II: effect of selected parameters on aggregation and    leakage. Int. J. Pharm. 146:193-204.-   9. Hong and McClements. 2007. Modulation of pH sensitivity of    surface charge and aggregation stability of protein-coated lipid    droplets by chitosan addition. Food Biophys. 2(1):46-55.-   10. Pallandre et al. November/December 2007. Improvement of    stability of oil-in-water emulsions containing caseinate-coated    droplets by addition of sodium alginate. J. Food Sci.    72(9):E518-E524.-   11. McClements. 2005. Theoretical analysis of factors affecting the    formation and stability of multilayered colloidal dispersions.    Langmuir 21(21):9777-85.-   12. Madrigal-Carballo et al. 2008. An approach to rheological and    electrokinetic behaviour of lipidic vesicles covered with chitosan    biopolymer. Colloids Surf., A 323:149-154.-   13. Madrigal-Carballo et al. 2009. Chitosomes loaded with cranberry    proanthocyanidins attenuate the bacterial lipopolysaccharide-induced    expression of iNOS and COX-2 in raw 264.7 macrophages. J. Liposome    Res. 19(3): 189-196.

Example 3 In Vitro Uptake Study of Protein-Loaded Chitosan-TanninNanoparticles as Adjuvants for Oral Vaccination

Novel vaccine adjuvants and particle-based delivery vehicles are beingevaluated in a variety of vaccines, including those against diseasessuch as cancer, malaria, AIDS, and hepatitis, among others [1], in whicha cellular and/or mucosal immune response is desired. The development ofsafe, novel adjuvants is necessary to maximize the efficacy of newand/or available vaccines. According to Gupta and Siber, an “ideal”adjuvant would elicit a persistent, high quality immune response to anantigen while being non-toxic, biodegradable, non-immunogenic, andchemically defined for reproducible manufacture [2].

Chitosan.

Chitosan is an abundant, natural linear polysaccharide derived by thedeacetylation of chitin from crustaceans, insects, and fungi [16, 17].Chitosan is non-toxic (LD₅₀>16 g/kg [18]), biodegradable [19],non-immunogenic [16], and can be manufactured reproducibly in accordancewith GMP guidelines. Chitosan's biodegradability, immunologicalactivity, and bioadhesion, make it an excellent candidate as adepot/adjuvant for vaccination.

Over 20 years ago, chitin derivatives, including chitosan, were found tobe potent activators of macrophages and NK cells [20, 21]. Thisimmunostimulating activity along with the structural similaritiesbetween chitin derivatives and glucans, an immunoadjuvant class ofnatural polysaccharides, led several scientists to study the adjuvantcapabilities of chitosan. Seferian and Martinez found that chitosanparticles, formulated in an emulsion with antigen, squalene andPluronic® L121, gave a prolonged, high antigen-specific antibody titerand sensitized animals for antigen-specific DTH responses following anIP injection.

Chitosan particles alone offered no enhancement of an adaptive immuneresponse [22]. However, because of its mucoadhesive properties, chitosanhas also been explored as an adjuvant for mucosal and subcutaneousvaccination [23]. Intranasal administrations of chitosan solutions haveenhanced adaptive immune responses to several antigens [24, 25]. Themechanisms of vaccine enhancement by chitosan are believed to be due toboth retention of vaccine in the nasal passages via mucoadhesion andopening of endothelial cell junctions for paracellular transport ofvaccine [25]. Recent clinical studies have confirmed that chitosan is apromising adjuvant platform for intranasal vaccination [26-28].

Due to the high protein binding properties of some types of chitosanmicroparticles, they are also potential candidates for oral delivery ofproteins and antigens [23]. Mild preparation can protect the proteinswhen they are incorporated during preparation of the microparticles [29,30]. In order to circumvent protein denaturation conditions, chitosanmicroparticles can be loaded passively [31].

Tannin.

Tannins are oligomeric polyphenolic plant compounds that complexproteins and polysaccharides. Consumption of foods, beverages andnutritional supplements that contain tannins is associated withdecreased risk of diseases which have an oxidative and microbialadherence etiology. However, the absorption of tannins from the gut islow. Greater than 95% of tannins are excreted in feces in complexes withproteins and polysaccharides from food or endogenous origins.

There are two groups of tannins: proanthocyanidins (PAC) andhydrolyzable tannins (HT). Examples of their chemical structures areshown in Schemes 1-4 above. Proanthocyanidins (PAC) are polymers offlavan-3-ols and flavans linked through an interflavan carbon bond, forexample between carbon 4 of the C ring and carbon 8 of the A ring.Hydrolyzable tannins are gallic acid and ellagic acid esters of coremolecules that consist of polyols such as sugars.

Chitosan-Tannin Nanoparticles.

By combining chitosan and tannin characteristics, specific, prolonged,and controlled release may be achieved [32]. Analysis of chitosan-tanninnanoparticles show that tannins increase rate of uptake of modelproteins and modulate subsequent T-cell responses, indicating thattannin-chitosan nanoparticles can affect antigen presentation in GALTdendritic cells and macrophages. This Example describes a system thatincludes of a unique combination of two carriers. The system has beenfound to achieve both stability and slow release of entrapped antigensand its effect on macrophage uptake and antigen presentation isdescribed.

Gut macrophages are maintained in a state of “inflammatory anergy” bycytokines, transforming growth factor β (TGF-β) and interleukin 10(IL-10), secreted by epithelial and stromal cells of the lamina propria[43, 44]. However, activated macrophages in GALT are associated withinflammatory bowl disease and colon cancer, and activated macrophages inthe oral cavity are associated with periodontal disease [39-42].

Tannins were isolated from cranberries (proanthocyanidins) andpomegranates (ellagitannins). The tannins were complexed to chitosanbiopolymers to prepare chitosan-tannin nanoparticles (CTNp) viaionotropic gelation with tripolyphosphate (TPP). The CTNp were loadedwith a model protein, such as bovine serum albumin (BSA), to determinethe effects of these CTNp on macrophage endocytosis. Cell culturemethods allowed for the determination of the effects of CTNp loaded withBSA, on macrophage uptake through microscopy of either fluorescentlylabeled biopolymer nor protein. Quenched BODIPY dye-labeled proteinsubstrates were used to study post endocytosis proteolysis of BSA-loadedCTNp by direct fluorescence measurement [35].

Materials and Reagents.

All reagents were at least analytical grade. Chitosan (deacetylationdegree of 92% calculated by ¹H NMR; mean molecular weight of 185 kDacalculated by specific viscosimetry) was provided by the PolymersResearch Laboratory (POLIUNA), National University, Costa Rica. Bovineserum albumin (BSA) [98% protein, Mw 66.3 kDa] and sodium triphosphatepentabasic (TPP) practical grade, 90-95%) were obtained fromSigma-Aldrich (St. Louis, Mo.) and were used without furtherpurification.

Cranberry PAC Fraction.

Spray dried cranberry juice powder was reconstituted in H₂O and appliedto a preparative LH-20 column equilibrated in water. Water was passedthrough the column to elute non-phenolic cranberry constituents. Aqueousacetone (4:1, acetone: H₂O, v:v) was then passed through the columnuntil it was white, to elute a crude cranberry PA fraction. The aqueousacetone fraction was concentrated by vacuum to remove the acetone. Itsgallic acid equivalent (GAE) was calculated by Folin-Ciocalteau assay(GAE=33.4 mg GAE/mL). The Cranberry PAC fraction was identified byMALDI-TOF MS as having a degree of polymerization (DP) ranging from 4 to7 with at least one A-type interflavan bond.

Pomegranate HT Fraction.

Nitrogen blended pomegranate peels (˜5 g) were extracted with aqueousacetone 80% v/v, filtered, and concentrated by vacuum. The concentratedpomegranate extract was applied to a C18 preparative column equilibratedwith water. Water was passed through the column to elute non-phenoliccranberry constituents. Aqueous methanol 50% v/v was then passed throughthe column until it was white, to elute a pomegranate HT fraction(GAE=4.13 mg GA/mL). MALDI-TOF MS and RP-HPLC analysis indicated thatthe pomegranate fraction was composed mainly of punicalagin (˜95%).

Preparation of Chitosan-Tannin Nanoparticles (CTNp).

Chitosan nanoparticles were prepared based on the ionotropic gelation ofchitosan with tripolyphosphate (TPP) anions. Chitosan was dissolved inacetic acid 1.00% v/v to obtain concentrations ranging 0.05-0.25% w/v.TPP was dissolved in water to a concentration of 1.00 mg/mL. Undermagnetic stirring at room temperature, 2 mL of TPP solution were addeddropwise to 5 mL of chitosan solution. The mixture was stirred for 60minutes followed by sonication.

Chitosan-tannin nanoparticles (CTNp) where obtained by mixing chitosannanoparticles (chitosan 0.10% w/v) with each tannin fraction indifferent volumetric ratios for 60 minutes at 25° C., as shown in Table3-1. After mixing, the resulting suspension was subsequently centrifugedat 12000×g for 15 minutes. The precipitate was suspended in water,centrifuged again, and then freeze-dried. The freeze-dried CTNp werethen resuspended in deionized water for further characterization andcell culture experiments.

TABLE 3-1 Composition of chitosan nanoparticles (CNp) andchitosan-tannin nanoparticles (CTNp) for delivery of antigenic hen-eggwhite lysozyme (HEL). Chitosan-TPP NP PAC fraction HT Fraction Sample ID(μL) (μL) (μL) CNp 0.05% 5000 0 0 CNp 0.10% 5000 0 0 CNp 0.25% 5000 0 0CPACNp 5% 4750 250 0 CPACNp 10% 4500 500 0 CPACNp 20% 4000 1000 0 CHTNp5% 4750 0 250 CHTNp 10% 4500 0 500 CHTNp 20% 4000 0 1000

Size and Zeta Potential.

Size determinations and electrophoretic mobility measurements werecarried out with a Z-Meter Zetasizer 2000 from Malvern Instruments, andwith a Z-Meter System 3.0 from Z-Meter USA equipped with microscopemodel DR from Carl Zeiss and an electrophoresis cell GT2 type. For sizeand electrophoretic mobility measurements, the samples were obtained asstated above and were afterwards diluted to 1:10 with deionized water.Five samples were prepared for each chitosan-tannin ratio. The error wasthe highest standard deviation for the five samples. For illustrativepurposes, approximate ζ-potential values were calculated starting fromthe Smoluchowski's equation, and using the following values:ε_(o)=8.9×10⁻¹² Fm⁻¹ and ε_(r)=79.

Thermal Analysis and Transmission Electron Microscopy.

A Perkin-Elmer DSC 7 differential scanning calorimeter (DSC) was used toevaluate the thermal properties of the chitosan and the chitosan-tanninnanoparticles, under N₂ atmosphere at a heating rate of 20 K/min from50° C. to 400° C. The nanostructure of CNp and CTNp was examined on aJEOL JSM-5200 transmission electron microscope (TEM) with a tilt angleof 30°.

Protein Loading and Release.

Bovine serum albumin (BSA) was used as a model protein. BSA loading ofCNp and CTNp was performed by incubating Np 10% v/v and BSA 0.5-2.5% w/vin phosphate buffered saline (PBS; pH 7.3) under shaking at 25° C. Afterincubation for 180 minutes, the suspension was centrifuged (1400 rpm for30 minutes) to remove the unloaded BSA. The loading degree wasdetermined by quantifying the non-bound BSA in the supernatant with theBradford protein assay. Both loading capacity (LC) and encapsulationefficacy (EE) were calculated as follows: LC=[(Total amount BSA−FreeBSA)/Nanoparticles weight] and EE=[(Total amount BSA−Free BSA)/TotalBSA].

BSA release from Np was determined in PBS (pH 7.3). To load the systems,5.0 mL of Np 10% v/v containing 0.5% (w/v) BSA was incubated for 3hours. After centrifuging (1400 rpm for 30 minutes) the loaded NP wereresuspended in PBS (pH 7.3) to make a 1.0% w/v suspension. Samples wereincubated at 37° C. under mild shaking After 15, 30, 45, 60, 90, 120,180, and 240 minutes, the tubes were given a spin-off and samples of 500μL of the supernatant were taken and replaced by 500 μL of PBS (pH 7.3).The non-bound HEL in PBS was determined with the Bradford protein assay.

Microscopy of Macrophage Endocytosis of BSA-Loaded CTNp.

RAW 264.7 macrophage-like cells were culture in 35 mm glass bottomculture plates (P35G-1.0-14-C, MatTek Corp., Ashland, Mass. 01721) andtreated with BSA and the BSA-loaded CNp and CTNp. Endocytosis of thenanosystems was studied by fluorescent microscopy of labeled BSA (AlexaFluor 488 Labeling Kit, Invitrogen/Molecular Probes, Eugene, Oreg.).Protein was labeled according to kit instructions and subsequentlyloaded into the Np according to the methodology described above.Macrophages were incubated with the labeled BSA and the BSA-loaded CNpand CTNp for 0.25-8 hours and imaged with a Zeiss fluorescent microscope(Carl Zeiss Microimaging, Thornwood, N.Y. 10594, with 450-490 nmexcitation and 510-565 nm emission filters).

Results and Discussion.

Characterization of Chitosan-Tannin Nanoparticles (CTNp).

Particle size is one of the most significant factors associated withmucosal and epithelial tissue uptake of nanoparticles and in theintracellular trafficking of the particles. Smaller size nanoparticles(˜100 nm) demonstrated more than 3-fold greater arterial uptake comparedto larger nanoparticles, as the smaller nanoparticles were able topenetrate throughout the sub-mucosal layers while the larger sizemicron-particles were predominantly localized in the epithelial lining[43]. The size and ζ-potential variations among the chitosan andchitosan-tannin nanoparticles, formulated at different chitosanconcentrations and chitosan to tannin volumetric ratios are shown inTable 3-2.

TABLE 3-2 Variation in size and ζ-potential for chitosan andchitosan-tannin nanoparticle formulations. Results are shown as mean ±standard error, n = 5. Sample ID Size (nm) ζ-Potential (mV) CNp 0.05%130 ± 1 19.6 ± 0.9 CNp 0.10% 149 ± 1 20.9 ± 1.9 CNp 0.25%  426 ± 28 32.2± 1.6 CHTNp 5% 165 ± 4 24.4 ± 0.5 CHTNp 10% 167 ± 3 22.8 ± 1.5 CHTNp 20%165 ± 4 22.6 ± 2.5 CPACNp 5% 298 ± 7 23.2 ± 3.1 CPACNp 10% 296 ± 2 21.5± 1.0 CPACNp 20% 292 ± 4 20.6 ± 0.9

Results show that both particle size and ζ-potential of the CNp increaseas a function of the biopolymer concentration, with a high increase ofabout 35% fold in size and 65% fold in ζ-potential, between the 0.10 andthe 0.25% w/v chitosan solutions. Meanwhile, CTNp prepared withpomegranate HT and cranberry PAC at a constant chitosan concentration of0.10% w/v show relatively constant values for the CHTNp prepared atincreasing volumetric ratios of HT (5-20% v/v). Each formulationincreased with the increasing concentration of polymer solution,indicating the formation of a coating layer on the surface of theliposome. The mean size of chitosan (0.5%)-coated liposomes was doublethat of the free liposomes.

Results showed an increase in the particle size as a function of theincrease in the chitosan concentration when preparing CNp. The mean sizeof the CNp formulated with chitosan 0.25 w/v was around 4 times largerthan those formulated with chitosan solutions below 0.10% w/v. CTNpshowed higher stability than CNp, indicating an effect of electrostaticstabilization driven by the chitosan-tannin interactions on the surfaceof the nanoparticles. The ζ-potential of the CNp was positive and wasdirectly proportional to the concentration of biopolymer. A ζ-potentialbelow ±30 mV is substantially necessary as a minimum for a physicalstable nanosuspension solely stabilized by electrostatic repulsion [44].The ζ-potential of the CNp showed values ranging 19 to 32 mV, whereasthe CTNp showed stable ζ-potential values of around +25 mV for bothCPACNp and CHTNp, confirming the effect of surface electrostaticstabilization.

The surface morphology of the CNp and CTNp was analyzed by ocularinspection of transmission electron microscopy (TEM) micrographs. Thenanoparticles were spherical in shape with a relatively homogeneous sizedistribution.

The DSC thermograms of CNp and CTNp (FIG. 18) presented an endothermicpeak at around 100° C., attributed to the evaporation of water absorbedthrough hydrogen bonding. This endothermic peak depends on thenanoparticle composition, showing, for instance, a shift to hightemperatures for the chitosan-tannin complexes, indicating an increasein hydrogen bonding driven by the increase in hydroxyl groups associatedwith the tannin moiety. An exothermic peak can be also observed for theCNp and CTNp at around 280° C., associated with chitosan degradation.This peak shows a shift to high temperatures when chitosan is complexedto tannins, indicating an increase in thermal stability due tochitosan-tannin interactions.

Protein Loading and Release.

The CNp were loaded with different amounts of bovine serum albumin (BSA)by incubation of CNp (chitosan 0.10% w/v) and CTNp (tannins 10% w/v)suspensions with 0.5-2.0% (w/v) BSA. Both encapsulation efficacy (LE)and loading capacity (LC) were determined. The data is illustrated inTable 3-3.

TABLE 3-3 Loading capacities and encapsulation efficiencies for loadedCNp and CTNp. Loading Capacity Encapsulation Efficacy BSA Conc. (% w/v)CNp CHTNp CPACNp CNp CHTNp CPACNp 0.50 61.2 ± 2.3 56.3 ± 1.7 52.2 ± 3.185.4 ± 3.6 86.9 ± 2.2 88.5 ± 2.4 1.00 58.7 ± 1.8 55.1 ± 1.3 51.5 ± 2.371.6 ± 2.7 79.8 ± 3.2 76.1 ± 2.7 1.50 60.3 ± 2.1 55.6 ± 1.9 52.7 ± 1.850.8 ± 2.4 60.1 ± 2.6 67.9 ± 3.1 2.00 56.2 ± 3.5 54.8 ± 2.0 51.9 ± 2.637.6 ± 3.1 44.5 ± 2.1 51.3 ± 2.9The CNp and CTNp particles were loaded with bovine serum albumin.Results are shown as mean±standard error, n=3.

Results indicated that the LC is not substantially influenced by theamount of BSA available in the loading solution, but CTNp appear to havelower loading capacities than CNp. The encapsulation efficacies of CNpseems to be increased when chitosan is complexed with tannins Thisproperty may be associated with the increase in hydrogen bondingpotential, as evidence by the DSC data. Therefore, BSA 1.0% w/v in theloading solution was selected as the optimal concentration. When CNp(0.10% w/v), CHTNp (10% w/v) and CPACNp (10% w/v) were incubated with1.0% of BSA, loading percentages of 58.7, 55.1 and 51.5 forindependently made batches were obtained, respectively. Under theseconditions, a very high EE of 71.6, 79.8 and 76.1 was obtained,indicating that only a small amount of BSA was lost during the loadingprocess.

Protein release studies showed that BSA total release at isothermalconditions, decreased significantly when the protein is delivered viaCTNp. FIG. 19 illustrates bovine serum albumin release profiles from CNpand CTNp. Addition of tannins provided a greater crosslinking densitywithin the biopolymer, causing increased packing and rigidity, as wellas increased inter-chain bonding, thereby reducing BSA release over timefrom the nanoparticulate system.

BSA labeled with fluorescein was loaded into chitosan nanoparticles withand without complexation to tannins The nanoparticles were incubatedwith Raw 264.7 macrophages. Uptake of the nanoparticulate systems wasfollowed by fluorescent microscopy. After 30 minutes, macrophagestreated with CTNp clearly contained more fluorescent endosomes than themacrophages treated with the CNp alone.

In vitro studies showed that CTNp increases macrophage uptake ofproteins, for example, BSA. Attenuation of gut macrophage activation byproteins loaded into CTNp is beneficial in treatment and prevention ofgut related infectious diseases. These new biomaterials can therefore beused as adjuvants, for example, for oral vaccination. Because uptake bymacrophage cells is the first step in vaccination, the results hereinindicate that chitosan-tannin nanoparticles can be used as efficientadjuvants for a vaccine delivery systems. While vaccines in generalprovoke stronger immune responses than proteins such as BSA, the CTNpsystem can be loaded with a variety of vaccines against differentpathologies.

In summary, chitosan binds to negatively charged tannins by anelectrostatic interaction driven by its positively charged amino groups.This interaction allows for the preparation of stable nanoparticles viaionotropic gelation with tripolyphosphate (TPP), suitable as a targetedcarrier and controlled release system for proteins, drugs and vaccines.The effect of chitosan-tannin nanoparticles (CTNp) on the uptake andrelease of bovine serum albumin (BSA) in Raw 264.7 macrophages wasstudied and described herein. CTNp were characterized according to size,zeta potential, and protein-loading and release properties. Resultsshowed an increase in the positive net charge and size of thenanoparticles as the concentration of chitosan was increased, indicatingan electrostatic interaction and a reliable covering, as determined byfluorescence microscopy. About 82% of the protein loaded remained in theCTNp after release studies for 4 hour in PBS. Confocal microscopystudies showed that CTNp had a higher uptake rate of the fluorescentlylabeled protein than chitosan nanoparticles without tannins (CNp) after30 minutes of incubation with the macrophages. Because uptake bymacrophage cells is the first step in oral vaccination, the CTNp systemcan be used as an oral vaccine delivery system.

Supporting information can be found in the following documents, whichare incorporated herein by reference.

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Example 4 Extraction of Cranberry Fruit Presscake to Obtain High Degreeof Polymerization Proanthocyanidins

The degree of polymerization, monomeric substitution, and nature ofintermolecular bonds are ‘proanthocyanidin variables’ that can becontrolled by choice of initial raw material (plants, fruits, juices),and extraction processes and enrichments by liquid and solid phasechromatography, including liquid/liquid (partition) chromatography andsupercritical fluid extraction. Research indicates that high degree ofpolymerization proanthocyanidins are more bioactive in in vitro modelsof: 1) inhibition of copper induced oxidation of low densitylipoprotein, 2) inhibition of E. coli invasion of mouse prostateepithelial cells, 3) inhibition of E. coli invasion of CaCo-2 cells, and4) inhibition of COX-2 and iNOS production by macrophages in response tolipopolysaccharide stimulus. This research indicates the value offormulations of tannin (PAC)-chitosan composites containing PAC ofhigher degree of polymerization.

Described herein is a method for extracting PAC of higher degree ofpolymerization from cranberry fruit presscake. Presscake is the materialthat remains after fruit has been subjected to the juicing process.While the process described here was applied to cranberries, the methodsdeveloped can be applied to any fruit or presscake to obtain PAC of withunique monomeric substitutions, interflavan bonds, and/or degrees ofpolymerization. The processes can therefore be used to recycle materialfrom juice processing waste streams by using them as source materialsfor tannin (PAC)-chitosan composites.

Methods.

Extraction of Cranberry Presscake.

Ten grams of cranberry presscake was homogenized in liquid nitrogen. Thepowdered material was extracted with 40 mL of aqueous acetone (70% v/v)in an ultrasonic bath for 15 minutes and then centrifuged at 2000 G for15 minutes. Supernatant was decanted and the extraction procedure wasrepeated two additional times on the remaining residue. Supernatantswere combined, acetone was removed by evaporation (<30° C.), and theextract was reconstituted in water (5 mL).

Proanthocyanidin Separation by Sephadex™ LH-20.

The extract of presscake was applied to a chromatography column (Kontes,2.5 cm ID×10 cm length) packed with Sephadex LH-20 (GE Healthcare,Uppsala, Sweden) equilibrated with water. Five fractions were obtainedby sequential elution with 100 mL of the following solvents: water(fraction 1) contained hydroxy-cinnamic acids and other non-phenoliccomponents; water:ethanol (1:1; fraction 2) contained primarilyanthocyanins; ethanol (fraction 3) contained primarily flavonols;ethanol:methanol (1:1; fraction 4) contained flavonol aglycones; andwater:acetone (1:4; fraction 5) contained PAC used in formulation oftannin-chitosan composites. Acetone was removed from Fraction 5 byevaporation (<30° C.), the extract was reconstituted with methanol to 5mL and used to produce tannin (PAC)-chitosan composites.

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight MassSpectrometry.

Mass spectra of proanthocyanidins were collected on a Bruker ReflexII-MALDI-TOF mass spectrometer (Billerica, Mass.) equipped with delayedextraction and a N2 laser (337 nm). In the positive reflectron mode anaccelerating voltage of 25.0 kV and a reflectron voltage of 26.5 kV wereused. In the positive linear mode and reflectron mode, an acceleratingvoltage of 25.0 kV was used. Spectra are the sum of 300 shots. Spectraare calibrated with bradykinin (1060.6 MW) and glucagon (3483.8 MW) asexternal standards. In accordance with previously published results[Krueger, 2000] trans-3-indoleacrylic acid (t-IAA; 5 mg/100 μl 80% aq.acetone) was used as a matrix. Dowex 50X8-400 cation exchange resin(Supelco), equilibrated in 80% aq. acetone (v/v) was used to deionizethe analyte:matrix solution.

Results.

MALDI-TOF mass spectral analysis of PAC obtained from cranberry juiceindicates that the degree of polymerization ranges from 2-7(catechin/epicatechin) units. MADLI-TOF mass spectral analysis of PACobtained from cranberry presscake indicates that the degree ofpolymerization ranges from 2-20 (catechin/epicatechin) units. FIG. 20illustrates positive mode MALDI-TOF MS [M+Na]⁺ of cranberryproanthocyanidins (PAC) from juice (top spectrum) and presscake (bottomspectrum). The juice PAC are oligomers from 2 to 7 degrees ofpolymerization (DP), whereas the presscake PAC are oligomers from 2 to13 DP in the main spectra and from 14 to 20 DP (m/z=5788) in the insert.

Therefore, the analysis herein shows that PAC of higher degree ofpolymerization are not effectively removed from the fruit during thejuicing process. A more aggressive extraction procedure (homogenizationof presscake and extraction in aqueous organic solvents) is required toobtain PAC of with high degrees of polymerization (i.e., greater than aDP of 7). Furthermore, subjecting the extract to chromatographicseparation on Sephadex LH20 resin affords a PAC fraction that was freefrom monomeric polyphenols (hydroxy cinnamic acids, anthocyanins, andflavonols).

Example 5 Non-Depectinized Cranberry Presscake Tannin (PAC)-Chitosan(Ch) Composites and Hydrogel: Agricultural Pathogenic Fungi(Colletotrichum acutatum)

Colletotrichum acutatum is a fruit rot pathogen that can affect mostplant parts (roots, leaves, blossoms, twigs and fruit). Most significantagricultural losses are due to infection of fruit causing disease infields (pre-harvest) and of the mature fruit (post-harvest) duringstorage (Wharton and Dieguez-Uribeonodo 2004).

This Examples shows that cranberry presscake tannin extract-chitosancomposites can inhibit germination of Colletotrichum acutatum conidia,and can inhibit the growth of Colletotrichum acutatum after conidiagermination.

Tannin-Chitosan Composite Formulations.

One gram of chitosan was dissolved in 1 L of 1% acetic acid (v/v) toproduce a 0.1% (w/v) stock solution. Five grams of cranberry presscake,obtained from the Ocean Spray Inc. Wisconsin Rapids Plant(non-depectinized line process #1), was homogenized in liquid nitrogen.The homogenized presscake was extracted with 20 mL of 70% aqueousacetone (v/v) in an ultrasonic bath for 10 minutes. The extract wascentrifuged at 2000 G for 10 minutes, and the supernatant was collected.The residual material was re-extracted two additional times as describedabove, and the supernatants were combined. Acetone was removed from thepresscake under reduced pressure (<30° C.). The extract was then appliedto a Sephadex LH-20 column (2.5×10 cm) that had been previouslyequilibrated in water. The column was sequentially eluted with water(100 mL), 50% aqueous ethanol (v/v; 100 mL) ethanol (100 mL), 50%ethanol:methanol (v/v; 100 mL) and 80% aqueous acetone (v/v; 300 mL).The 80% aq. Acetone fraction was concentrated by roto-vacuum evaporationto a concentration of 29.15 mg Gallic acid equivalents/mL, as determinedby the Folin-Ciocalteau assay. MALDI-TOF mass spectrometry characterizedthe cranberry presscake PAC extract as having 2-20 degree ofpolymerization (see FIG. 20).

A cranberry presscake tannin extract (Pac)-Chitosan (Ch) composite wasprepared by mixing 5 mL of a 0.1% chitosan solution with 3.43 mL of the29.15 mg GAE/mL presscake extract. The final composite was 20% Pac and80% Ch by weight. The composite solution was lyophilized andreconstituted in water the same day as the experiments described below.

Colletotrichum Condidial Germination and Growth Assay.

Culture Plates:

-   -   1) Minimal Media (MM); defined media—10 mL in 60 mm plate;        Gel-rite.    -   2) Potato Dextrose Agar (PDA); undefined media—10 mL in 60 mm        plate.    -   3) Blueberry Agar (BB); undefined media—10 mL in 60 mm plate.

Colletotrichum is a significant pathogen of blueberry fruit.Accordingly, an undefined blueberry media as a nutrient source wasdeveloped. Blueberry fruits were homogenized, autoclaved, and mixed withagar.

After the culture plates cooled, 1 mL of PacCh (20:80, w/w) compositesolution was added at 0, 40, 200 and 1000 GAE (gallic acid equivalents).The plates were swirled to allow for equal distribution of the compositesolution on the surface. The plates were allowed to dry under a laminarflow hood. A thin hydrogel (film) was generally formed with minimalcomposite solution being absorbed into the media. After the plates dried(and films formed), 5 μL of a Colletotrichum acutatum condidialsuspension was pipetted into the center of each plate. Plates were thenmonitored for conidial germination, fungi growth, and morphologychanges.

Minimal Media Results.

After 4 days of incubation, there was no significant difference inculture diameter, however differences in culture morphology wereapparent. Higher GAE plates showed less coloring and appeared lessmature.

Potato Dextrose Agar Results.

After 4 days of incubation, significant difference in both culturediameter and culture morphology were apparent. Higher GAE plates showedless coloring and appeared less mature. In one experiment, at Day 4, theculture diameter of the 0 GAE plate was 21 mm, while the culturediameter of the 1000 GAE plate was only 16 mm. The culture of the 1000GAE plate also had less coloring and appeared significantly less mature.

Blueberry Agar Results.

After 4 days of incubation, significant differences in both culturediameter and culture morphology were apparent. The area of the culturesof higher GAE plates were significantly smaller. The area of the cultureof the 1000 GAE plate appeared to be less then 10% of the area of the 0GAE plate, as determined by ocular inspection. Additionally, germinationin the 1000 GAE plate sample was delayed until Day 3 of incubation.

Example 6 Tannin-Chitosan Composites Inhibit E. Coli Invasion of Caco-2Cells

Colonization of intestinal epithelial cells by pathogenic bacteria, suchas E. coli, cause intestinal diseases, including diarrhea and urinarytract infections (UTIs). The three specific pathogenic strains of E.coli that are significant causes of diarrhea in infants and travelers toareas with poor sanitation are the enterotoxigenic (ETEC),enteropathogenic (EPEC), and enteroinvasive (EIEC) strains.

Infant mortality from an outbreak of E. coli induced diarrhea can havefatality rates as high as 40%. The Centers for Disease Control andPrevention have classified E. coli 0157:H7 as one of the more virulentstrains, reporting that 20,000 cases of infection may occur annually. E.coli 0157:H7 is found in the intestinal tract of cattle. Improperhandling and preparation (e.g., undercooking) of ground beef is aleading cause of human infection.

Urinary tract infections are a major medical concern for women and canrecur frequently. The vast majority of UTIs are caused by E. colibacteria ascending through the urethra, attaching to the walls of thebladder, multiplying, and causing infection. Antibiotics are prescribedfor mild cases, while severe cases often require hospitalization. Thereis growing concern about uropathogenic bacterial resistance toantibiotics used for UTI treatment. Cranberry proanthocyanidins can actby inhibiting P-fimbriated uropathogenic strains of E. coli fromadhering to uroepithelial cells, which is the initial step indevelopment of infection. The new compositions and methods describedherein can be used for treating diarrhea and UTIs.

Tannin-Chitosan Composite Formulations.

The tannin-chitosan composite formulations were prepared as describedabove in Example 5. The composite solutions were either prepared freshor lyophilized and reconstituted in water the day of the experimentsdescribed below.

Tannin-Chitosan Composites Inhibit E. coli Adhesion to Caco-2 Cells.

Caco-2 cells are a human cell line derived from a carcinoma of the colonthat exhibits structural and functional differentiation patternscharacteristic of mature enterocytes in post-confluent cultures. Caco-2cells were grown in DMEM+10% FBS+1% dipeptide glutamine+1% Penn/Strep+1%Non-essential Amino Acids and were plated in 24-well cell cultureplates. One week after confluency, the cells are differentiated and weresubsequently used for the anti-invasion study. After numerous cellcounting, the Caco-2 cells averaged 800,000 cells per well on the day ofthe experiment.

Six replications for each treatment were performed. E. coli strain 5011was thawed and grown in tryptose broth for 72 hours prior to the startof the experiment, including one passage after 48 hours. Theconcentration of bacteria was determined by reading a dilution at 420nm, which was repeatedly 9×10⁹ bacteria per mL. A ratio of 100 bacteriaper Caco-2 cell was used for the study.

The bacteria and each of the three treatment compositions: cranberrypresscake tannin (5 μg gallic acid equivalent), chitosan (25 μg) andtannin-chitosan (5 μg gallic acid equivalent: 25 μg chitosan), wereallowed to react for 10 minutes at room temperature prior to theaddition of the experimental cell culture medium (RPMI-1640 plus HEPESwith 5% FBS). Caco-2 cells were washed twice with 1×PBS. The bacteriaand treatments were further incubated in the medium for 10 minutes priorto being added to the Caco-2 cells. Cells were incubated for 1 hour at37 degrees C. Cells were washed once in PBS, then incubated at 37degrees C. for 1 hour with a 100 ug/mL Gentamicin solution (made inRPMI/HEPES plus 5% FBS) to kill any extracellular bacteria. Cells werewashed twice with PBS, then incubated for 30 minutes at room temperaturewith a 1× Triton solution (made in PBS) to lyse the Caco-2 cells. Thiscellular extract was plated onto Eosin Methylene Blue Agar plates aswhole extract, 1:10, and 1:100 dilutions. Plates were inverted andincubated overnight at 37 degrees C. Colony forming units were countedthe next day. Colony forming units represent the number of E. coli thatinvaded the Caco-2 cells.

Results.

Test results indicated that cranberry presscake tannin (5 μg gallic acidequivalent), chitosan (25 μg) and tannin-chitosan (5 μg gallic acidequivalent: 25 μg chitosan) composite treatments all inhibited invasionof Caco-2 cells by E. coli strain 5011. Cranberry presscake tanninsalone were the most bio-active, followed by the composite material.Chitosan alone was the least effective. The variability between freshlyprepared composite material and lyophilized composites was within thevariability of replication. FIG. 21 illustrates the results of theexperiments.

Example 7 Tannin-Chitosan Composite and Nano-Particle Synthesis

This example describes how the amount of tannin (cranberry presscakePAC) and amount of 3-polyphosphate affect particle size. The followingprocedures were used.

1.) A 5 mg/mL Chitosan Stock Solution was prepared by dissolving 5 gramsof chitosan into 1000 mL with 1% acetic acid solution.

2.) A 1 mg/mL Chitosan Solution was prepared. DI water was used todilute 20 mL of the 5 mg/mL Chitosan solution into 100 mL.

3.) The Stock Cranberry Presscake was diluted to create a PAC WorkingStock. Five grams of cranberry presscake was then processed andchromatographed as described above in Example 5. A cranberry presscaketannin extract (Pac)-Chitosan (Ch) composite was made by mixing 5 mL ofa 0.1% chitosan solution with 3.43 mL of the 29.15 mg GAE/mL presscakeextract. The final composite was 20% Pac and 80% Ch by weight. Thecomposite solutions were either prepared fresh or lyophilized andreconstituted in water the day of the experiment. 0.5 mL of stockcranberry presscake (29.15 mg Gallic Acid/mL) was diluted into 5.0 mLwith DI water to yield a 2.915 mg GA/mL PAC Working Stock.

4.) Composite Solutions were prepared. Five 5 mL of 1 mg/mL Chitosansolution was pipetted into fifteen 10 mL beakers each (solutions 1-15).All solutions were then stirred vigorously. Constant stir rates weremaintained; 0.343 mL (1.0 mg) of PAC Working Stock was pipetted intosolutions 1-5, 0.257 mL (0.75 mg) into solutions 6-10, and 0.1715 mL(0.5 mg) into solutions 11-15. Stirring was continued for 15 additionalminutes.

5.) Nanoparticles were then prepared. Varying volumes of TPP solution (1mg/mL) were quickly (not dropwise) pipetted into each solution. TPPvolumes of 0.25 mL, 0.50 mL, 0.75 mL, 1.00 mL, and 1.25 mL were used forsolutions 1-5, 6-10, and 11-15, respectively. An appropriate volume ofDI water was added to each solution in order for all the solutions tohave a total volume of 7 mL. Stirring was continued for 15 additionalminutes.

6.) Nanoparticle size was then measured. The parameters of the particlesize instrument were set to 5 runs at 30 seconds each and a RealRefractive Index of 1.420.

7.) The Nanoparticle Samples were then Lyophilized, re-suspend, andmeasured. The nanoparticle solutions were placed into a freezer untilfrozen and then placed into a freeze-dryer for 48 hours. The dry sampleswere re-suspended with DI water to 7.0 mL. Samples were mixed untilsuspension was complete. The re-suspended nanoparticles were measured atfive 30 second runs and a Real Refractive Index of 1.420.

TABLE 7-1 PAC-Ch Composite Nanoparticle Preparation and CompositionData. Sample Composition PAC Working PAC/Chitosan TPP/Chitosan ChitosanStock TPP Sample Ratio Ratio (1 mg/mL) (2.915 mg/mL) (1 mg/mL) DI Water1 1:5 1:20 5.0 mL 0.343 mL 0.25 mL 1.407 mL (5.0 mg) (1.00 mg) (0.25 mg)2 1:5 1:10 5.0 mL 0.343 mL 0.50 mL 1.157 mL (5.0 mg) (1.00 mg) (0.50 mg)3 1:5 1:6.67 5.0 mL 0.343 mL 0.75 mL 0.907 mL (5.0 mg) (1.00 mg) (0.75mg) 4 1:5 1:5 5.0 mL 0.343 mL 1.00 mL 0.657 mL (5.0 mg) (1.00 mg) (1.00mg) 5 1:5 1:4 5.0 mL 0.343 mL 1.25 mL 0.407 mL (5.0 mg) (1.00 mg) (1.25mg) 6 1:6.67 1:20 5.0 mL 0.257 mL 0.25 mL 1.493 mL (5.0 mg) (0.75 mg)(0.25 mg) 7 1:6.67 1:10 5.0 mL 0.257 mL 0.50 mL 1.243 mL (5.0 mg) (0.75mg) (0.50 mg) 8 1:6.67 1:6.67 5.0 mL 0.257 mL 0.75 mL 0.993 mL (5.0 mg)(0.75 mg) (0.75 mg) 9 1:6.67 1:5 5.0 mL 0.257 mL 1.00 mL 0.743 mL (5.0mg) (0.75 mg) (1.00 mg) 10 1:6.67 1:4 5.0 mL 0.257 mL 1.25 mL 0.493 mL(5.0 mg) (0.75 mg) (1.25 mg) 11 1:10 1:20 5.0 mL 0.1715 mL 0.25 mL 1.579mL (5.0 mg) (0.50 mg) (0.25 mg) 12 1:10 1:10 5.0 mL 0.1715 mL 0.50 mL1.329 mL (5.0 mg) (0.50 mg) (0.50 mg) 13 1:10 1:6.67 5.0 mL 0.1715 mL0.75 mL 1.079 mL (5.0 mg) (0.50 mg) (0.75 mg) 14 1:10 1:5 5.0 mL 0.1715mL 1.00 mL 0.829 mL (5.0 mg) (0.50 mg) (1.00 mg) 15 1:10 1:4 5.0 mL0.1715 mL 1.25 mL 0.579 mL (5.0 mg) (0.50 mg) (1.25 mg)Results.

TABLE 7-2 Data used to prepare FIG. 22. BATCH 1 Jun. 3, 2009 Sample IDSize (nm) SE (±) PAC/Chito 1:10; TPP 1:20 895.0 85.8 PAC/Chito 1:10; TPP1:10 653.4 24.7 PAC/Chito 1:10; TPP 1:6.67 512.7 28.7 PAC/Chito 1:10;TPP 1:5 439.6 25.4 PAC/Chito 1:10; TPP 1:4 321.1 13.7 PAC/Chito 1:6.67;TPP 1:20 692.6 39.8 PAC/Chito 1:6.67; TPP 1:10 568.9 48.6 PAC/Chito1:6.67; TPP 1:6.67 481.2 36.3 PAC/Chito 1:6.67; TPP 1:5 414.3 27.6PAC/Chito 1:6.67; TPP 1:4 272.0 7.5 PAC/Chito 1:5; TPP 1:20 559.1 20.1PAC/Chito 1:5; TPP 1:10 429.5 12.0 PAC/Chito 1:5; TPP 1:6.67 362.1 21.3PAC/Chito 1:5; TPP 1:5 306.8 10.4 PAC/Chito 1:5; TPP 1:4 235.3 10.0

TABLE 7-3 Data used to prepare FIG. 23. BATCH 2 Jun. 4, 2009 Sample IDSize (nm) SE (±) PAC/Chito 1:10; TPP 1:20 718.1 64.1 PAC/Chito 1:10; TPP1:10 486.5 13.1 PAC/Chito 1:10; TPP 1:6.67 367.3 7.5 PAC/Chito 1:10; TPP1:5 305.5 15.8 PAC/Chito 1:10; TPP 1:4 239.4 4.4 PAC/Chito 1:6.67; TPP1:20 608.7 21.1 PAC/Chito 1:6.67; TPP 1:10 446.1 9.1 PAC/Chito 1:6.67;TPP 1:6.67 341.6 11.1 PAC/Chito 1:6.67; TPP 1:5 303.7 9.3 PAC/Chito1:6.67; TPP 1:4 217.5 4 PAC/Chito 1:5; TPP 1:20 481.1 17 PAC/Chito 1:5;TPP 1:10 365.6 13.3 PAC/Chito 1:5; TPP 1:6.67 293.2 6.9 PAC/Chito 1:5;TPP 1:5 246.5 2.2 PAC/Chito 1:5; TPP 1:4 203.8 4.4

TABLE 7-4 Data used to prepare FIG. 24. BATCH 2 LYOPHILIZED ANDRE-SUSPENDED Jun. 17, 2009 Sample ID Size (nm) SE (±) PAC/Chito 1:10;TPP 1:20 2885.3 146.0 PAC/Chito 1:10; TPP 1:10 4309.8 720.4 PAC/Chito1:10; TPP 1:6.67 2955.4 369.0 PAC/Chito 1:10; TPP 1:5 2388.2 332.5PAC/Chito 1:10; TPP 1:4 1396.7 193.4 PAC/Chito 1:6.67; TPP 1:20 4446.4267.9 PAC/Chito 1:6.67; TPP 1:10 2414.0 468.0 PAC/Chito 1:6.67; TPP1:6.67 2314.5 467.3 PAC/Chito 1:6.67; TPP 1:5 2016.9 64.7 PAC/Chito1:6.67; TPP 1:4 1300.5 181.6 PAC/Chito 1:5; TPP 1:20 1470.0 26.3PAC/Chito 1:5; TPP 1:10 1701.9 318.0 PAC/Chito 1:5; TPP 1:6.67 1505.382.9 PAC/Chito 1:5; TPP 1:5 1366.7 342.4 PAC/Chito 1:5; TPP 1:4 689.746.2

Summary.

TPP and PAC concentrations both affect particle size. TPP concentrationand particle size share an inverse relationship. At all threeproanthocyanidin concentrations tested, an increase in TPP concentrationreduced particle size. Increasing the PAC concentration also reducesparticle size. The graphic results indicate a logarithmic trend ofincreasing TPP concentration versus particle size. As PAC concentrationscontinue to increase beyond 1:5, there is a point at which increased PACconcentrations ceases to cause a decrease in particle size. Likewise,there is a maximum limit of how high the TPP concentration can be withcontinued decreases in particle size. The re-suspended particle datashows that the nanoparticles become unstable and may fall into solutionafter a period of seven days of refrigerated storage (˜37° C.).

Example 8 Grape Seed Tannins-Chitosan Composite and Nano-ParticleSynthesis

Determinations were made regarding how the amount of tannins in grapeseed extract (GSE) and the amount of 3-polyphosphate affect particlesize. The GSE-chitosan nanoparticles have similar physical and chemicalcharacteristics to cranberry tannin-chitosan composites regardingparticle size and the effect of the tannin to chitosan loading. Thefollowing procedure was used for the Grape Seed Extract (GSE)/ChitosanNanoparticle preparation.

1.) A 5 mg/mL Chitosan Stock Solution was prepared. Five 5 grams ofchitosan was dissolved into 1000 mL DI water with 1% acetic acidsolution. The solution was vacuum filter with a Whatman #41 filter.

2.) A 1 mg/mL Chitosan Solution was prepared. DI water was used todilute 20 mL of 5 mg/mL Chitosan solution into 100 mL.

3-1.) Stock Grape Seed Extract was diluted to create a GSE WorkingStock. 0.2835 mL of stock Grape Seed Extract (51.41 mg Gallic Acid/mL)was diluted into 5.0 mL with DI water to yield a 2.915 mg GA/mL GSEWorking Stock.

4.) Composite solutions were then prepared. Five mL of 1 mg/mL Chitosansolution was pipetted into fifteen 10 mL beakers each (solutions 1-15).The solutions were vigorously stirred. Constant stir rates weremaintained and 0.343 mL (1.0 mg) of GSE Working Stock was pipetted intosolutions 1-5, 0.257 mL (0.75 mg) was pipetted into solutions 6-10, and0.1715 mL (0.5 mg) was pipetted into solutions 11-15. Stirring wascontinued for 15 minutes.

5.) Nanoparticles were then prepared. Varying volumes of TPP solution (1mg/mL) were quickly pipetted into each solution. TPP volumes of 0.25 mL,0.50 mL, 0.75 mL, 1.00 mL, and 1.25 mL were used for solutions 1-5,6-10, and 11-15. An appropriate volume of DI water was added to eachsolution in order for all the solutions to have a total volume of 7 mL.The solutions were stirred for 15 additional minutes.

6.) Nanoparticle size was measured. The parameters of the particle sizeinstrument were set to 3 runs at 30 seconds each and a Real RefractiveIndex of 1.420.

7.) The process was repeated using cranberry proanthocyanidins.

3-2.) Stock cranberry PAC was diluted to create a PAC Working Stock.0.2445 mL of stock cranberry PAC's from Batch 3 (59.61 mg GallicAcid/mL) was diluted into 5.0 mL with DI water to yield a 2.915 mg GA/mLPAC Working Stock.

Preparation data and results are provided in Tables 8-1 and 8-2 and theresults are illustrated in FIGS. 25 and 26. FIG. 25 illustrates thegrape seed extract (GSE)/chitosan nanoparticle size distribution, andFIG. 26 illustrates a dose-response, showing the effects of GSE andchitosan-GSE composites on bacterial invasion of Caco-2 cells. Theeffects (mean±SD) of Grape Seed Extract (GSE) Tannins, and Chitosan-GSEcomposite nanoparticles on the invasion of Caco-2 cells by E. colistrain 5011 are clearly demonstrated. An asterisk indicates an effectthat is statistically significant compared to the corresponding control.This data indicates that the Chitosan-GSE composite nanoparticles aresignificantly more active than GSE alone.

TABLE 8-1 Procedure Chart. Sample Comp. GSE&PAC/ TPP/ GSE&PAC ChitosanChitosan Chitosan Working Stock TPP Sample Ratio Ratio (1 mg/mL) (2.915mg/mL) (1 mg/mL) DI Water 1 1 to 5 1 to 20 5.0 mL 0.343 mL 0.25 mL1.4069 mL (5.0 mg) (1.00 mg) (0.25 mg) 2 1 to 5 1 to 10 5.0 mL 0.343 mL0.50 mL 1.1570 mL (5.0 mg) (1.00 mg) (0.50 mg) 3 1 to 5 1 to 6.67 5.0 mL0.343 mL 0.75 mL 0.9069 mL (5.0 mg) (1.00 mg) (0.75 mg) 4 1 to 5 1 to 55.0 mL 0.343 mL 1.00 mL 0.6569 mL (5.0 mg) (1.00 mg) (1.00 mg) 5 1 to 51 to 4 5.0 mL 0.343 mL 1.25 mL 0.4069 mL (5.0 mg) (1.00 mg) (1.25 mg) 61 to 6.67 1 to 20 5.0 mL 0.257 mL 0.25 mL 1.4927 mL (5.0 mg) (0.75 mg)(0.25 mg) 7 1 to 6.67 1 to 10 5.0 mL 0.257 mL 0.50 mL 1.2427 mL (5.0 mg)(0.75 mg) (0.50 mg) 8 1 to 6.67 1 to 6.67 5.0 mL 0.257 mL 0.75 mL 0.9927mL (5.0 mg) (0.75 mg) (0.75 mg) 9 1 to 6.67 1 to 5 5.0 mL 0.257 mL 1.00mL 0.7427 mL (5.0 mg) (0.75 mg) (1.00 mg) 10 1 to 6.67 1 to 4 5.0 mL0.257 mL 1.25 mL 0.4927 mL (5.0 mg) (0.75 mg) (1.25 mg) 11 1 to 10 1 to20 5.0 mL 0.1715 mL 0.25 mL 1.5785 mL (5.0 mg) (0.50 mg) (0.25 mg) 12 1to 10 1 to 10 5.0 mL 0.1715 mL 0.50 mL 1.3285 mL (5.0 mg) (0.50 mg)(0.50 mg) 13 1 to 10 1 to 6.67 5.0 mL 0.1715 mL 0.75 mL 1.0785 mL (5.0mg) (0.50 mg) (0.75 mg) 14 1 to 10 1 to 5 5.0 mL 0.1715 mL 1.00 mL0.8285 mL (5.0 mg) (0.50 mg) (1.00 mg) 15 1 to 10 1 to 4 5.0 mL 0.1715mL 1.25 mL 0.5785 mL (5.0 mg) (0.50 mg) (1.25 mg)

TABLE 8-2 Summary of Average Particle Size. Sample ID Run 1 Run 2 Run 3Average SE (±) GSE/Chito 1:5; 749.9 701.6 729.1 726.9 14 TPP/Chito 1:20GSE/Chito 1:5; 603.4 613.8 600.9 606 3.9 TPP/Chito 1:10 GSE/Chito 1:5;492.3 511.8 514.5 506.2 7 TPP/Chito 1:6.67 GSE/Chito 1:5; 402.3 392.3408.2 401 4.7 TPP/Chito 1:5 GSE/Chito 1:5; 302.5 314.1 301.8 306.1 4TPP/Chito 1:4 GSE/Chito 1:6.67; 653.2 628.6 631.7 637.8 7.8 TPP/Chito1:20 GSE/Chito 1:6.67; 512.5 525.1 494.2 510.6 9 TPP/Chito 1:10GSE/Chito 1:6.67; 447.3 440.3 443.8 443.8 2 TPP/Chito 1:6.67 GSE/Chito1:6.67; 346.9 356.4 341.5 348.3 4.4 TPP/Chito 1:5 GSE/Chito 1:6.67;274.7 275.6 266.8 272.4 2.8 TPP/Chito 1:4 GSE/Chito 1:10; 679.1 601.4654.6 645 22.9 TPP/Chito 1:20 GSE/Chito 1:10; 498.7 539.1 553.6 530.516.4 TPP/Chito 1:10 GSE/Chito 1:10; 502.9 471.4 447.7 474 16 TPP/Chito1:6.67 GSE/Chito 1:10; 322.9 343.2 319.5 328.5 7.4 TPP/Chito 1:5GSE/Chito 1:10; 262.9 279.9 270.6 271.1 4.9 TPP/Chito 1:4

Example 9 Tannin-Chitosan Composites as Therapeutic Biomaterials

This example demonstrates the application of tannin-chitosan compositenanoparticles for use as therapeutic biomaterials to control pathogenicmicrobial colonization of human epithelial cells. The tannin-chitosancomposite nanoparticles can therefore be used for the treatment andprevention of human disease states such as, diarrhea, resultant ofenterotoxigenic Escherichia coli (ETEC) and other pathogenic microbecolonization of the intestinal epithelial cells, and urinary tractinfections (UTI) resulting from adhesion of p-fimbriated uropathogenicbacteria to uroepithelial cells.

Results show that tannin-chitosan composite nanoparticles significantlyreduced invasion of intestinal epithelial cells by uropathogenic E.coli. Furthermore, scanning electron microscopy allowed for theidentification of a putative mechanism by which invasion is inhibited.Tannin-chitosan composite nanoparticles are believed to coat andcross-link E. coli flagella, thereby inhibiting invasion.

Results.

Invasion of Intestinal Epithelial Cells.

Using an assay that developed to study the invasion of gastrointestinal(GI) epithelial cells (Caco-2) by uropathogenic Escherichia coli (UPEC),the relative invasiveness of 30 strains of UPEC isolated from women withchronic UTI was examined (FIG. 27). The relative ability of the UPECstrains to invade GI epithelial cells correlated with their ability toinvade uroepithelial cells (prostate cells) in culture (data not shown).Of the strains, UPEC 5011 was found to be most invasive (FIG. 27). Forthis reason UPEC 5011 was used in all experiments.

Cranberry Tannin-Chitosan Composite Nano-Particles Inhibit Invasion ofIntestinal Epithelial Cells.

We have developed LC and MALDI-TOF mass spectrometric techniques tofirst separate tannins from other polyphenolic compounds andsubsequently characterize the structural heterogeneity and oligomericdistribution of tannins. In these experiments, tannins were extractedfrom cranberry press cake and 1) used alone or 2) combined with chitosanto form nano-particles composite materials. The tannins alone, chitosannanoparticles alone and tannin-chitosan composite nanoparticles werethen mixed with UPEC 5011 before incubation with Caco-2 cells.

Results of a dose-response experiment indicate the tannin-compositematerial significantly reduced invasion of epithelial cells at a lowerdose than the tannin perpetration alone. The cranberry tanninpreparation alone significantly inhibited the ability of the pathogen toinvade the intestinal epithelial cells by ˜82% at a total polyphenolicconcentration (0.5 μg of gallic acid equivalence (GAE)/mL and by ˜96% at0.75 μg GAE/mL (FIG. 28). The cranberry tannin-composite nanoparticlessignificantly inhibited the ability of the pathogen to invade theintestinal epithelial cells by ˜40% at a total polyphenolicconcentration of 0.2 μg GAE/mL, by ˜80% at 0.5 μg GAE/mL and by ˜96% at0.75 μg GAE/mL (FIG. 28).

The results illustrated in FIG. 29 shows the effects (mean±SD) ofchitosan nanoparticles, cranberry tannins, and cranberry tannin-chitosancomposite nanoparticles on the invasion of Caco-2 cells by E. colistrain 5011. An asterisk indicates an effect that is statisticallysignificant compared to the corresponding control. Dose is 0.75 uggallic acid equivalent. The results indicate that chitosan nanoparticlesalone were not significantly different from controls in preventing UPEC5011 invasion of intestinal epithelial cells.

Additional comparisons of tannin preparations with varying polymerlength indicated that a higher degree of inhibition of invasiveness wasachieved by tannins with greater degree of polymerization (DP).Cranberry tannin are believed to inhibit invasion by binding to thepathogen and disrupting the surface adhesion molecules required forinvasion. Thus a tannin with greater DP is likely to have a greateraffinity for the pathogen. The effects of this interaction was examinedusing scanning electron microscopy (SEM).

Effect of Cranberry Tannin-Chitosan Composite Nanoparticles on thePhysical Structure UPEC Flagella.

FIG. 30 shows scanning electron micrographs exploring the effect ofchitosan nanoparticles and tannin-chitosan composite nanoparticles onUPEC 5011 flagella structure and subsequent impact on invasion ofintestinal epithelial cells in vitro. The arrow in Panel A shows thenormal physical structure of flagella expressed by UPEC 5011 insuspension.

When the pathogen was exposed to a chitosan nanoparticle preparation, nodisruption of the normal structure of flagella is observed (Panel B).When the pathogen was exposed to the tannin-chitosan compositenanoparticle material, extensive coating and cross-linking of flagellaon multiple cells is seen (Panel C). It was also noted that thisinteraction created numerous aggregates of UPEC. Panel D is a scanningelectron micrograph of the tannin-chitosan composite nanoparticle alone.These results indicate that the cranberry tannin-chitosan compositematerials physically coat the flagella of UPEC, which in turn preventsinvasion of the intestinal epithelial cell.

Data has also been obtained indicating that composites can affectbacterial fimbriae, thereby restricting bacterial locomotion byphysically preventing the flagella from providing motion.Tannin-chitosan composites can also affect cell membrane integrity byinhibiting the production and expression of lipopolysaccharides.

Protocol for Preparation of CaCo2 Cells and E. coli Strain 5011 forScanning Electron Microscopy.

Bacterial and Cell Preparation.

CaCo2 cells were cultured according to standard laboratory protocol.Sterile cover slips were placed into 12 wells of a 24-well plate. CaCo2cells were seeded in each well 10 days before the experiment. Medium waschanged every 48 hours. Three days prior to the experiment a static,aerobic culture of E. coli (strain 5011) was grown at 37° C. in 40 mL ofTryptose broth for 48 hours. One milliliter of bacteria was passed fromthe top of the broth into 40 mL of Tryptose broth and incubatedovernight under the above conditions.

Day of Experiment.

The overnight bacterial culture was centrifuged at 1840×g for 10minutes, washed twice in PBS, and was re-suspended in a final volume of1 mL PBS. The optical density at 450 nm of a 1/100 dilution of thebacterial suspension was determined, then compared to a previouslygenerated growth curve. CaCo2 cells were washed twice with PBS.

Bacteria were used at a multiplicity of infection (MOI) of 100. Bacteriaand cranberry products were mixed in a 15 mL conical tube and allowed toincubate at room temperature for 5 minutes. Cell culture medium wasadded to the bacteria and cranberry products, and divided amongst theappropriate wells of CaCo2 cells, if applicable. Cells are incubated for1 hour at 37° C. (no CO₂ or humidity control), then washed once in PBS.Cell culture medium plus Gentamicin sulfate (100 m/mL finalconcentration) was added to the plate incubated for 1 hour at 37° C. (noCO₂ or humidity control). Cells were washed three times in PBS beforeSEM preparation.

SEM Preparation.

Cells were fixed in the 24-well plate overnight at 4° C. using a 2%Gluteraldehyde solution in 0.1M Phosphate buffer. Cells were washed in0.1M phosphate buffer for 10 minutes. Cover slips were removed from thewells and stacked into a holder with washers between the cover slips topreserve the surface. Cells were washed again in the holder with 0.1Mphosphate buffer for 10 minutes. Cells were subsequently washed inincreasing concentrations of ethanol for 10 minutes each, according tothe following sequence:

Percent EtOH Time (minutes) 30 10 50 10 70 10 80 10 90 10 95 10 100  10100  10 Siv-dried 10The holder was placed in the critical point dryer chamber with ethanol.The dryer was cooled to 10° C. using CO₂. The ethanol was purged andreplaced with CO₂, then incubated for 10 minutes. Two additional purgesand incubations were performed. The chamber was then heated to 35°C.-42° C., increasing the pressure on the samples. The pressure was thenslowly decreased (˜100 psi/min) until reaching 0 psi. Samples wereremoved from the holder and placed in a desiccator until coated andobserved on a scanning electron microscope.

Materials.

Tryptose Broth Recipe:

10 g Tryptose (Fisher #211713);

2.5 g NaCl (Fisher #BP358-212);

0.5 g Dextrose (Fisher #215530);

0.0025 g Thiamine Hydrochloride (Sigma #T4625-10 g).

Autoclave on the liquid cycle at 121° C. and 15 psi; no dry time.

Experimental Cell Culture Medium:

RPMI-1640 (Fisher #SH30255.FS);

Fetal Bovine Serum (Fisher #SH3007003). FBS is 5% total medium (v/v).

CaCo2 Standard Protocol Cell Culture Medium:

DMEM (Fisher #BW12614F);

Fetal Bovine Serum (Fisher #SH3007003). FBS is 10% total medium (v/v);

Penicillin/Streptomycin Mix (Fisher #AT35712). Pen/Strep is 1% totalmedium (v/v);

Nonessential Amino Acids (NEAA; Fisher #13-114E); NEAA is 1% totalmedium (v/v);

Gluta-MAX (L-alanyl-L-glutamine, Invitrogen #35050-061). Gluta-MAX is 1%of the total medium (v/v).

Example 10 Pharmaceutical Dosage Forms

The following formulations illustrates representative pharmaceuticaldosages forms that may be used for the therapeutic or prophylacticadministration of a tannin-chitosan composition described herein, suchas a nanoparticle or liposome (hereinafter referred to as ‘CompositionX’):

(i) Tablet 1 mg/tablet ‘Composition X’ 100.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 300.0

(ii) Tablet 2 mg/tablet ‘Composition X’ 20.0 Microcrystalline cellulose410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0500.0

(iii) Capsule mg/capsule ‘Composition X’ 10.0 Colloidal silicon dioxide1.5 Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0600.0

(iv) Injection 1 (1 mg/mL) mg/mL ‘Composition X’ 1.0 Dibasic sodiumphosphate 12.0 Monobasic sodium phosphate 0.7 Sodium chloride 4.5 1.0NSodium hydroxide solution q.s. (pH adjustment to 7.0-7.5) Water forinjection q.s. ad 1 mL

(v) Injection 2 (10 mg/mL) mg/mL ‘Composition X’ 10.0 Monobasic sodiumphosphate 0.3 Dibasic sodium phosphate 1.1 Polyethylene glycol 400 200.001N Sodium hydroxide solution q.s. (pH adjustment to 7.0-7.5) Water forinjection q.s. ad 1 mL

(vi) Aerosol mg/can ‘Composition X’ 20.0 Oleic acid 10.0Trichloromonofluoromethane 5,000.0 Dichlorodifluoromethane 10,000.0Dichlorotetrafluoroethane 5,000.0

These formulations may be prepared by conventional procedures well knownin the pharmaceutical art. It will be appreciated that the abovepharmaceutical compositions may be varied according to well-knownpharmaceutical techniques to accommodate differing amounts and types ofactive ingredient ‘Composition X’. Aerosol formulation (vi) may be usedin conjunction with a standard, metered dose aerosol dispenser.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. A composition comprising a matrix of chitosan andtannins, wherein the tannins comprise one or more oligomericproanthocyanidins, the chitosan is electrostatically bonded to the oneor more oligomeric proanthocyanidins, and the matrix of chitosan andtannins is in the form of a chitosan-tannin composite material, whereinthe composition is a hydrogel film, the chitosan-tannin compositematerial does not include a crosslinking agent, and the tensile strengthof the composite material is higher than that of the same hydrogel filmcontaining chitosan but no oligomeric proanthocyanidin.
 2. Thecomposition of claim 1 wherein the tensile strength of the compositematerial is 700 MPa to about 825 MPa.
 3. The composition of claim 2 incombination with a wound dressing.
 4. The composition of claim 2 incombination with a dermal patch.
 5. The composition of claim 2 incombination with a packaging biomaterial.
 6. A composition comprising amatrix of chitosan and tannins, wherein the tannins comprise one or moreoligomeric proanthocyanidins, the chitosan is electrostatically bondedto the one or more oligomeric proanthocyanidins, and the matrix ofchitosan and tannins is in the form of a chitosan-tannin compositematerial, wherein the composition is in the form of a hydrogel film or abiogel, the chitosan-tannin composite material does not include acrosslinking agent, and the tensile strength of the composite materialis about 600 MPa to about 825 MPa.
 7. The composition of claim 6 incombination with a wound dressing.
 8. The composition of claim 6 incombination with a dermal patch.
 9. The composition of claim 6 incombination with a packaging biomaterial.